Micacocidin derivatives

ABSTRACT

The object of the present invention is to provide a novel compound which has various biological activities and is useful for medical and animal drugs. The present invention provides a compound represented by the formula: ##STR1## wherein R 1  is COOR 4 , CONR 5  R 6 , CO--R 7  --OR or CH 2  OR 8  ; R 2  is hydrogen atom, alkyl, aralkyl, heteroaryl, heteroarylalkyl, COR 13 , COOR 14 , CONR 15  R 16  ; R 3  is hydrogen atom or OR 3  ; a broken line ( - - - ) represents the presence of a double bond when R 3  is oxygen atom and the absence of a double bond when R 3  is OR 3 , or a salt or metal chelete thereof.

DETAILED DESCRIPTION OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a novel compound useful in the field ofmedicine and veterinary medicine, and to medical and animal drugscontaining said compound.

BACKGROUND OF THE INVENTION

Pyochelin, which is produced by Pseudomonas aeruginosa, is known as acompound having a similar structure to that of the present invention (J.Bacteriology 137(1), 357, 1979). However, it is unknown whether it hasalso a biological activity similar to that of the compound of thepresent invention.

Subject of the Invention to be Solved

The object of the present invention is to provide a novel compoundhaving various biological activities useful for medical and animaldrugs.

Means for Solving the Subject

The present inventors have made extended studies to attain the objectmentioned above and have found that a novel compound of the formula (I)shown below has anti-mycoplasma activity, anti-coccidium activity,antibacterial activity, antifungal activity and immunosuppressiveactivity.

Thus, the present invention provides a compound of the formula (I):##STR2## wherein R¹ is (1) COOR⁴ wherein R⁴ is hydrogen atom, optionallysubstituted alkyl, optionally substituted aralkyl, optionallysubstituted heteroaryl or optionally substituted heteroarylalkyl; (2)CONR⁵ R⁶ wherein R⁵ and R⁶ are the same or different and representhydrogen atom, hydroxyl, optionally substituted alkyl, optionallysubstituted alkoxyl, optionally substituted aryl, optionally substitutedaralkyl, optionally substituted heteroaryl or optionally substitutedheteroarylalkyl, provided that when one of R⁵ and R⁶ is hydroxyl oroptionally substituted alkoxyl, the other is hydrogen atom, optionallysubstituted alkyl, optionally substituted aryl or optionally substitutedaralkyl; (3) CO--R⁷ --OR wherein R⁷ is α-amino acid residue and R ishydrogen atom or alkyl ; or (4) CH₂ OR⁸ wherein R⁸ is hydrogen atom,optionally substituted alkyl, optionally substituted aralkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl, COR⁹wherein R⁹ is hydrogen atom, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl or optionally substituted heteroarylalkyl, COOR¹⁰ wherein R¹⁰is optionally substituted alkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl or optionallysubstituted heteroarylalkyl, or CONR¹¹ R¹² wherein R¹¹ and R¹² are thesame or different and represent hydrogen atom, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl or optionally substitutedheteroarylalkyl;

R² is hydrogen atom, optionally substituted alkyl, optionallysubstituted aralkyl, optionally substituted heteroaryl, optionallysubstituted heteroarylalkyl, COR¹³ wherein R¹³ is hydrogen atom,optionally substituted alkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl or optionallysubstituted heteroarylalkyl, COOR¹⁴ wherein R¹⁴ is optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedaralkyl, optionally substituted heteroaryl or optionally substitutedheteroarylalkyl, or CONR¹⁵ R¹⁶ wherein R¹⁵ and R¹⁶ are the same ordifferent and represent hydrogen atom, optionally substituted alkyl,optionally substituted aryl, optionally substituted aralkyl, optionallysubstituted heteroaryl or optionally substituted heteroarylalkyl;

a broken line (- - - ) represents the presence or absence of a doublebond, provided that when it presents the presence of the double bond, R³is oxygen atom, or when it represents the absence of the double bond, R³is OR^(3') wherein R ^(3') is hydrogen atom, optionally substitutedalkyl, optionally substituted aralkyl, optionally substitutedheteroaryl, optionally substituted heteroarylalkyl, COR ¹⁷ wherein R¹⁷is hydrogen atom, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted heteroarylor optionally substituted heteroarylalkyl, COOR¹⁸ wherein R¹⁸ isoptionally substituted alkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl or optionallysubstituted heteroarylalkyl, or CONR¹⁹ R²⁰ wherein R¹⁹ and R²⁰ are thesame or different and represent hydrogen atom, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl or optionally substitutedheteroarylalkyl, or

a salt or metal chelate thereof, provided that the metal chelate isexcluded when R¹ is COOH, R² is hydrogen atom, and

R³ is OH.

An optionally substituted alkyl represented by R², R⁴, R⁵, R⁶, R⁸, R⁹,R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R^(3')includes C₁ -C₁₀ alkyl, preferably C₁ -C₄ alkyl, specifically methyl,ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl andthe like, preferably methyl, ethyl and propyl, and especially preferablymethyl.

When these alkyls are substituted, the substituents include halogen atom(for example, fluorine, chlorine, bromine and iodine); C₁ -C₅ alkoxy,preferably C₁ -C₃ alkoxy (for example, methoxy, ethoxy and propoxy); C₁-C₅ alkylthio, preferably C₁ -C₃ alkylthio, for example, methylthio,ethylthio or propylthio; alkoxycarbonyl having C₁ -C₅ alkoxyl,preferably C₁ -C₃ alkoxyl, for example, methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl or the like; carboxy; hydroxy; hydroxy; amino which isoptionally substituted with C₁ -C₅ alkyl or alkanoyl, preferably C₁ -C₃alkyl or alkanoyl, for example, amino, monomethyl amino, dimethyl amino,acethyl amino or the like; or C₁ -C₃ alkanoyloxy, for example, acetoxylor the like, preferably halogen atom, alkoxy or hydroxy, andparticularly chlorine, fluorine, methoxy, ethoxy or hydroxy ispreferred.

These substitutents can bind to said alkyl at any possible position, andthe number of the substituents is preferably one to three, morepreferably one to two.

An optionally substituted aralkyl represented by R², R⁴, R⁵, R⁶, R⁸, R⁹,R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R^(3')includes for example benzyl, phenethyl, (α- or β-) naphthyl methyl andthe like. Possible substituents on the aralkyl group include halogenatom, for example, fluorine, chlorine, bromine or iodine; C₁ -C₅ alkyl,preferably C₁ -C₃ alkyl, for example, methyl, ethyl, propyl and thelike; C₁ -C₅ alkoxy, preferably C₁ -C₃ alkoxy, for example, methoxy,ethoxy, propoxy and the like; C₁ -C₅ alkyl halide, preferably C₁ -C₃alkyl halide, for example, fluoromethyl, dichloromethyl, difluoromethyl,trifluoromethyl, fluoroethyl, dichloroethyl and the like; nitro; cyanoand so on. These substitutents may bind to any possible position on thearalkyl group, and the number of the substituents is preferably one tothree, more preferably one to two.

An optionally substituted heteroaryl represented by R², R⁴, R⁵, R⁶, R⁸,R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R^(3')include the heteroaryl containing in its ring one or more oxygen atoms,sulfur atoms or nitrogen atoms arbitrarily selected, for example,pyridyl, pyrimidinyl, furyl, thienyl, pyrrolyl, quinolyl, oxazolyl,isoxazolyl, indolyl and the like. The heteroaryl may be optionallysubstituted, by halogen atom, for example, fluorine, chlorine, bromineor iodine atom; C₁ -C₅ alkyl, preferably C₁ -C₃ alkyl, for example,methyl, ethyl, propyl and the like; C₁ -C₅ alkoxy, preferably C₁ -C₃alkoxy, for example, methoxy, ethoxy, propoxy and the like; C₁ -C₅ alkylhalide, preferably C₁ -C₃ alkyl halide, for example, fluoromethyl,dichloromethyl, difluoromethyl, trifluoromethyl, fluoroethyl,dichloroethyl and the like; nitro; cyano and so on. These substitutentsmay bind to any possible position on the heteroaryl ring, and the numberof the substituents is preferably one to three, more preferably one totwo. The heteroaryl may further be fused with other heteroaryl or carbonring.

Examples of optionally substituted heteroarylalkyls represented by R²,R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹,R²⁰ and R^(3') include those which are composed of the heteroarylmentioned above and C₁ -C₅ alkyl, preferably C₁ -C₂ alkyl, for example,methyl, ethyl or the like. Specific examples are picolyl, pyridylethyl,thenyl, fulfuryl and the like. The heteroarylalkyls may be substitutedby similar substitutents to those aforementioned for the heteroarylgroup.

An optionally substituted alkoxy represented by R⁵ and R⁶ includes C₁-C₅ alkoxy, preferably C₁ -C₃ alkoxy, and specific examples are methoxy,ethoxy, propoxy, isopropoxy and the like. The alkoxy may be substitutedby halogen atom, for example, fluorine, chlorine, bromine and iodineatom; C₁ -C₅ alkoxy, preferably C₁ -C₃ alkoxy, such as methoxy, ethoxy,propoxy and the like; and optionally substituted phenyl, such aschlorophenyl, tolyl, methoxyphenyl, nitrophenyl, and the like. Thesesubstitutents may bind to any possible position on the alkoxy group, andthe number of the substituents is preferably one to three, morepreferably one to two.

Examples of the optionally substituted aryl represented by R⁵, R⁶, R⁹,R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ include C₆ -C₁₄aryl, preferably C₆ -C₁₀ aryl. Specific examples are phenyl, α-naphthyl,β-naphthyl and the like. The aryl is optionally substituted by a halogenatom, for example, fluorine, chlorine, bromine and iodine; C₁ -C₅ alkyl,preferably C₁ -C₃ alkyl, for example, methyl, ethyl, propyl and thelike; C₁ -C₅ alkoxy, preferably C₁ -C₃ alkoxy, for example, methoxy,ethoxy, propoxy and the like; C₁ -C₅ alkyl halide, preferably C₁ -C₃alkyl halide, for example, fluoromethyl, dichloromethyl, difluoromethyl,trifluoromethyl, fluoroethyl, dichloroethyl and the like; nitro; cyanoand the like.

These substitutents may bind to any possible position of said aryl, andthe number of the subusutituents is preferably one to three, morepreferably one to two.

An α-amino acid residue represented by R⁷ includes cysteine (Cys)residue, glycine (Gly) residue, serine (Ser) residue, alanine (Ala)residue and the like.

An alkyl represented by R includes the same groups as those exemplifiedas the alkyl groups of the optionally substituted alkyl represented byR² or the like stated above.

R¹ is preferably the groups represented by COOR⁴ wherein R⁴ ispreferably hydrogen, optionally substituted alkyl or optionallysubstituted benzyl, or CONR⁵ R wherein R⁵ and R⁶ are independentlypreferably hydrogen atom, hydroxy, alkoxy, alkyl, carboxyalkyl,alkoxycarbonylalkyl or hydroxyalkyl. More preferably, R¹ is carboxy,alkoxycarbonyl, alkoxyalkoxycarbonyl, hydroxyalkoxycarbonyl,benzyloxycarbonyl, benzyloxycarbonyl halide, carbamoyl,monoalkylcarbamoyl, dialkylcarbamoyl, hydroxycarbamoyl,N-alkylhydroxycarbamoyl, carboxyalkylcarbamoyl oralkoxycarbonylalkylcarbamoyl.

R² is preferably hydrogen atom, optionally substituted alkyl, optionallysubstituted benzyl or optionally substituted alkanoyl. More preferably,R² is hydrogen atom, alkyl, alkoxyalkyl, alkyl halide, benzyl halide, oralkanoyl.

R³ is preferably oxygen atom or a group represented by OR^(3') whereinR^(3') is preferably hydrogen atom, optionally substituted alkyl,optionally substituted benzyl or optionally substituted alkanoyl. Morepreferably, R³ is oxygen atom, alkoxy, alkoxyalkoxy, alkoxy halide,benzyloxy halide or alkanoyloxy.

A method of preparing the compounds of the present invention is asfollows. ##STR3##

The compound of the present invention (I-1) can be prepared by reactingmicacocidin with an acid in an appropriate solvent.

The acid which can be used includes hydrochloric acid, sulfuric acid,potassium hydrogensulfate, and methane sulfonate. The acid is used in anamount of 2-50 equivalents, preferably 3-10 equivalents of micacocidin.

The solvent which can be used includes carbohydrate halides, such asdichloromethane, chloroform and the like; esters, such as ethyl acetateand the like; ethers, such as diethylether, tetrahydrofuran and thelike; alcohols, such as methanol, ethanol and the like; water; andmixtures thereof.

The reaction temperature is -20 to 50° C., preferably 0 to 35° C.

The reaction time is 1 minute to 1 hour, preferably 5 minutes to 0.5hour.

The compound of the present invention (1-1) obtained can be purified bya conventional method, for example, column chromatography,recrystalization or the like.

Micacocidin used as a starting material in the reaction of Scheme 1 canbe obtained as a culture product of a microorganism belonging to GenusPseudomonas, for example, Pseudomonas sp. strain No. 57-250.

Cultivation of Pseudomonas sp. strain No. 57-250 can be performed in aliquid medium consisting of several compositions. A medium useful forproducing micacocidin contains as a carbon source, for example, glucose,and as a nitrogen source, for example, yeast extract, soy bean powder,pharmamedia and the like. In addition, a metal salt such as zinc,copper, iron, or calcium carbonate is added for preparing the medium, ifnecessary. An antifoaming agent, such as polypropylene glycol can beadded, if necessary. Cultivation is usually performed aerobically,preferably aeration culture with stirring. The cultivation temperaturecan suitably be changed in the range in which the microorganism can growto produce micacocidin. However, especially preferable temperature is23-28° C. The pH is preferably near 7, and the cultivation time isusually about 24 to 48 hours, and then, cultivation is discontinued at aproper time when the titer of micacocidin in the culture reaches tomaximum level.

Recovery of micacocidin from the culture medium may be performedaccording to conventional methods for recovering fermentation products,for example, by extracting the product with non-hydrophilic organicsolvents such as ethyl acetate, chloroform or the like, or by adsorbingthe culture filtrate to synthetic partitioned porous resin (HP-20) orthe like, which is then eluted with methanol or ethanol, and then,concentrating the eluate thus obtained in vacuo, followed by adding anorganic solvent such as ethyl acetate to extract the product.

Micacocidin thus obtained can be used in the subsecuent steps withoutperforming further purification, or after purified by the combination ofwell known methods usually used for purification of lipophilicsubstances, for example, thin layer chromatography and columnchromatography using a carrier such as silica gel, or columnchromatography using molecular sieve (LH-20 etc.), followed bycrystallizing micacocidin by precipitating it from various mixedsolvents, such as methanol-water, ethanol-water, methanol-ethyl acetateor the like.

Bacteriological property of Pseudomonas sp. strain No. 57-250 is shownbelow.

1. Morphology:

It is Gram-negative rod, and 0.5-0.6 μm×2.0-3.0 μm in size.

It has motility with one or more polar flagella.

2. Findings in Culture:

1) Culture in broth medium

A bit slow growth of bacteria, observed a slight yellowish whiteprecipitate.

2) Stab culture in broth agar

Observed growth in thread form along the stab line with broad growthappeared on the surface of the medium.

No production of gas and pigment was observed.

3) Slant culture in broth agar

A bit slow growth of bacteria. Bacterial cells are yellowish white, dullglossy and wet. Flat swelling. Transparency: translucent in an earlystage of culture, and then turned to opaque with the lapse of time. Noproduction of gas and pigment was observed.

4) Cultivation on broth agar plate medium:

A bit slow growth of bacteria. Colony: translucent and yellowish-whitesmall dot and thereafter growing into opaque and yellowish-white roundform with flat or round swelling. No production of gas and solublepigment was observed.

3. Physiological and Biochemical Properties

(1) Requirement of oxygen: Yes

(2) Optimal temperature for growth: 30° C. (well growing at 28° C. and37° C., slow growing at 10° C., and not growing at 4° C. and 41° C.)

(3) Optimal pH for growth: pH 7 (grew at pH 5-8, but not grew at pH 4and pH 9)

(4) Denitrification: negative

(5) Reduction of nitrate: negative

(6) Oxidase test: positive

(7) Urease test: positive

(8) Catalase test: positive (weakly)

(9) Liquefaction of gelatin: negative

(10) Starch degradation: negative

(11) Methyl red test: negative

(12) V-P test: negative

(13) Production of indole: negative

(14) Production of H2S: negative

(15) Availability of citric acid: positive (Christensen medium andSimons medium)

(16) Hydrolizing ability for Tween 80: positive (slow)

(17) Hydrolizing ability for escrine: negative

(18) Hydrolizing ability for arginine: negative

(19) Decarboxylation of lysin: negative

(20) Decarboxylation of ornithine: negative

(21) β-galactosidase: negative

(22) Coagulation of milk: negative

(23) Peptonization of milk: negative

(24) O-F test: oxidized (slow)

(25) Accumulation of PHB: negative

(26) Quinone type: Q8

(27) Fluorescent pigment: negative

(28) Acid-producing ability from sugar:

An acid is produced from D-glucose, D-galactose and D-xylose, and notproduced from D-fructose, maltose, D-mannitol, lactose, sucrose,D-arabinose, D-sorbitol, D-mannose and D-trehalose.

(29) Assimilating ability of carbon sources

It can assimilate as a single carbon source D-glucose, D-galactose,D-xylose, inositol succinate and lactic acid to form the bacterial cellsthereof.

On the other hand, it did not assimilate D-fructose, maltose,D-mannitol, lactose, sucrose, D-arabinose, D-sorbitol, D-mannose andD-trehalose, geraniol, L-valine, β-alanine, DL-arginine, betaine andmethanol.

The growth of strain No. 57-250 in an inorganic salt medium containing asole carbon source is very slow and weak.

In view of these results, the present bacteria are considered to belongto Pseudomonas. Therefore, their properties were further compared withthose of Pseudomonas species described in Bergey's Manual of SystematicBacteriology Vol.1, and it can be found that no species have propertiesconsistent or analogous to those of the present bacteria. Thus, Thebacteria was designated as Pseudomonas sp. No.57-250.

The strain was deposited under accession No. FERM P-14235 with NationalInstitute of Bioscience and Human Technology, Higashi 1-1-3, Tsukuba,Ibaraki, JAPAN on Mar. 17, 1994 and then transferred to theInternational Deposition under Budapest Treaty on Jun. 22, 1995, andassigned to accession No. FERM BP-5143. ##STR4## wherein R²² representsoptionally substituted alkyl, optionally substituted aralkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl, COR¹⁷herein R¹⁷ is as defined above, COOR¹⁸ wherein R¹ is as defined above,or CONR¹⁹ R²⁰ wherein R¹⁹ and R²⁰ are as defined above.

Out of the compounds of the present invention represented by the formula(I-2), the compound wherein R²² is optionally substituted alkyl,optionally substituted aralkyl, optionally substituted heteroaryl oroptionally substituted heteroarylalkyl can be prepared by alkylating orarylating micacocidin in an appropriate solvent in the presence orabsence of a base, and then, if necessary, allowing to react with anacid. An alkylating agent or arylating agent includes, for example,diazoalkanes such as diazomethane, trimethylsilyldiazomethane or thelike; alkyl halides such as iodomethane, bromomethane, benzyl bromide orthe like; heteroaryl halides such as 2-chloropyridine, 4-chloropyridineor the like; heteroarylalkyl halides such as2-(chloromethyl)benzoimidazol or the like; sulfates such as dimethylsulfate, diethyl sulfate or the like; sulfonates such as ethyl methanesulfonate, phenethyl p-toluene sulfonate or the like; andchloromethylalkyl ethers such as chloromethylmethyl ether,chloromethylethyl ether or the like. They are used in an amount of 1-5equivalents, preferably 1-3 equivalents of micacocidin.

A solvent which can be used in the reaction includes alcohols such asmethanol, ethanol or the like; carbohydrate halides such asdichloromethane, chloroform, chlorobenzene or the like; esters such asethyl acetate or the like; ethers such as diethyl ether,tetrahydrofuran, dioxane or the like; amides such asN,N-dimethylformamide, N,N-dimethylacetoamide or the like; sulfoxidessuch as dimethyl sulfoxide or the like; carbohydrates such as hexane,benzene, toluene or the like; ketones such as acetone, ethyl methylketone or the like; nitriles such as acetonitrile or the like; water;and a mixure thereof.

Bases to be employed include carbonates, for example, potassiumcarbonate, sodium carbonate and the like; alkali hydroxides, forexample, potassium hydroxide, sodium hydroxide and the like; and amines,for example, triethyl amine, N,N-diisopropylethylamine and the like.They are used in an amount of 1-5 equivalents, preferably 1-3equivalents of micacocidin.

The reaction temperature is -20 to 120° C., preferably 0 to 60° C., andthe reaction time is 10 min. to 24 hrs., preferably 30 min. to 15 hrs.

The reaction with acids can be performed in the same manner as thereaction in Scheme 1 described above.

Out of the present compounds of the formula (I-2), the compound whereinR²² is COR¹⁷ can be prepared by acylating micacocidin in an appropriatesolvent in the presence or absence of a base, and then, reacting theproduct with an acid if necessary.

Acylating agents include, for example, acid anhydrides such as aceticanhydride, benzoic anhydride and the like; acid halides, such as acethylchloride, benzoyl chloride, phenylacethyl chloride and the like. Theyare used in an amount of 1-5 equivalents, preferably 1-3 equivalents ofmicacocidin.

Solvents to be used include carbohydrate halides such dichloromethane,chloroform, chlorobenzene and the like; esters such as ethyl acetate andthe like; ethers such as diethyl ether, tetrahydrofurane, dioxane andthe like; amides such as N,N-dimethylformamide, N,N-dimethylacetoamideand the like; sulfoxides such as dimethyl sulfoxide; carbohydrates suchas hexane, benzene, toluene; ketones such as acetone, ethyl methylketone; nitriles such as acetonitrile; and the mixture thereof.

Bases to be used include carbonates such as potassium carbonate, sodiumcarbonate and the like; amines such as triethylamine,N,N-diisopropylethylamine and the like. The bases are used in an amountof 1-5 equivalents, preferably 1-3 equivalents of micacocidin.

The reaction temperature is -20 to 60° C., preferably 0 to 40° C., andthe reaction time is 5 minutes to 24 hours, preferably 10 minutes to 8hours.

The reaction with an acid can be performed in the same manner as thereaction in Scheme 1 previously stated. out of the present compounds ofthe formula (I-2), the compound wherein R²² is COOR¹⁸ can be prepared,in the presence or absence of a base, by carbonating micacocidin in anappropriate solvent, and then, reacting the product with an acid ifnecessary. The carbonating agents include, for example, halogenatedcarbonates such as ethyl chlorocarbonate, t-butyl bromocarbonate, phenylchlorocarbonate, benzyl cholorocarbonate and the like. They are used inan amount of 1-5 equivalents, preferably 1-3 equivalents of micacocidin.

Solvents to be used include carbohydrate halides such asdichloromethane, chloroform, chlorobenzene and the like; esters such asethyl acetate and the like; ethers such as diethyl ether,tetrahydrofurane, dioxane and the like; amides such asN,N-dimethylformamide, N,N-dimethyacetoamide and the like; sulfoxidessuch as dimethyl sulfoxide; carbohydrates such as hexane, benzene,toluene; ketones such as acetone, ethyl methyl ketone; nitriles such asacetonitrile; and a mixture thereof.

Bases to be used include carbonates such as potassium carbonate, sodiumcarbonate; amines such as triethylamine, N,N-diisopropylethylamine. Theyare used in an amount of 1-5 equivalents, preferably 1-3 equivalents ofmicacocidin.

The reaction temperature is -20 to 60° C., preferably 0 to 40° C., andthe reaction time is 5 minutes to 24 hours, preferably 10 minutes to 8hours.

Reaction of the product with an acid can be performed in the same manneras the reaction in Scheme 1 stated above.

Out of the compounds of the formula (I-2) of the present invention, thecompound wherein R²² is COR¹⁹ R²⁰ can be prepared, in the presence of orthe absence of a base, by carbamoyling micacocidin in an appropriatesolvent, and then, reacting the product with an acid, if necessary.

The carbamoyling agents include, for example, isocyanates such as methylisocyanate, phenyl isocyanate, benzyl isocyanate and the like; carbamylhalides such as dimethylcarbamyl chloride and the like. They are used inan amount of 1-5 equivalents, preferably 1-3 equivalents of micacocidin.

Solvents to be used include halogenated carbohydrates such asdichloromethane, chloroform, chlorobenzene and the like; esters such asethyl acetate and the like; ethers such as diethyl ether,tetrahydrofurane, dioxane and the like; amides such asN,N-dimethylformamide, N,N-dimethylacetoamide and the like; sulfoxidessuch as dimethyl sulfoxide; carbohydrates such as hexane, benzene,toluene and the like; ketones such as acetone, ethyl methyl ketone;nitrites such as acetonitrile; and a mixture thereof.

Bases to be used include carbonates such as potassium carbonate, sodiumcarbonate and the like; amines such as triethylamine,N,N-diisopropylethylamine and the like. They are used in an amount of1-5 equivalents, preferably 1-3 equivalents of micacocidin.

The reaction temperature is -20 to 60° C., preferably 0 to 40° C., andthe reaction time is 5 minutes to 24 hours, preferably 10 minutes to 8hours.

The reaction of the product with an acid can be performed in the samemanner as the reaction described in Scheme 1.

The compound (I-2) of the present invention thus obtained can bepurified by conventional methods such as column chromatography,recrystallization and the like, if necessary. ##STR5## wherein R²¹ isoptionally substituted alkyl, optionally substituted aralkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl, COR¹³wherein R¹³ is as defined above, COOR¹⁴ wherein R¹⁴ is as defined above,or CONR¹⁵ R¹⁶ wherein R¹⁵ and R¹⁶ are as defined above.

Out of the compounds of the formula (I-3) of the present invention, thecompound wherein R²¹ is optionally substituted alkyl, optionallysubstituted aralkyl, optionally substituted heteroaryl or optionallysubstituted heteroarylalkyl, can be prepared, in the presence or absenceof a base, by alkylating or arylating the compound of the presentinvention in an appropriate solvent, followed by hydrolyzing the productwith a base in an appropriate solvent, if necessary.

The alkylation or arylation can be performed in the same manner as thereaction described in Schema 2. Bases to be used in the hydrolysisinclude carbonates such as potassium carbonate, sodium carbonate and thelike; alkali hydroxides such as sodium hydroxide, potassium hydroxideand the like. They are used in an amount of 1-3 equivalents, preferably1-2 equivalents of the compound (I-1).

Solvents to be used include alcohols such as methanol, ethanol and thelike; ethers such as diethyl ether, tetrahydrofuran, dioxane and thelike; carbohydrates such as hexane, benzene, toluene and the like;ketones such as acetone, ethyl methyl ketone and the like; water; and amixture thereof.

The reaction temperature is -20 to 120° C., preferably 0 to 40° C., andthe reaction time is 10 minutes to 24 hours, preferably 30 minutes to 15hours.

Out of the compounds of the formula (I-3) of the present invention, thecompound wherein R²¹ is COR¹³ can be prepared, in the presence orabsence of a base, by acylating the compound (I-1) of the presentinvention in an appropriate solvent.

Acylation can be performed in the same manner as the reaction in Scheme2.

Out of the present compounds of the formula (I-3), the compound whereinR²¹ is COOR¹⁴ can be prepared, in the presence or absence of a base, bycarbamoyling the compound (I-1) of the present invention in anappropriate solvent.

Carbonation can be performed in the same manner as the reaction inScheme 2.

Out of the present compounds of the formula (I-3), the compound whereinR²¹ is CONR¹⁵ R¹⁶ can be prepared, in the presence or absence of a base,by carbamoyling the compound (I-1) of the present invention in anappropriate solvent.

Carbamoylation can be performed in the same manner as the reaction inScheme 2.

The compound (I-3) of the invention thus obtained can be optionallypurified by a conventional method, for example, column chromatograpy,recrystalization and the like. ##STR6## wherein R²¹ and R²² are the sameor different and as defined above.

Out of the compounds of the formula (I-4) of the present invention, thecompound wherein R²¹ and R²² are the same and represent optionallysubstituted alkyl, optionally substituted aralkyl, optionallysubstituted heteroaryl or optionally substituted heteroarylalkyl, can beprepared, in the presence or absence of a base, by alkylating orarylating micacocidine in an appropriate solvent.

Alkylation or arylation can be performed in the same manner as thereaction described in Scheme 2. Out of the present compounds of theformula (I-4), the compound wherein R²¹ is COR¹³ and R²² is COR¹⁷, canbe prepared, in the presence or absence of a base by acylatingmicacocidin in an appropriate solvent.

Acylation can be performed in the same manner as the reaction in Scheme2 stated above.

Out of the present compounds of the formula (I-4), the compound whereinR²¹ is COOR¹⁴ and R²² is COOR¹⁸ can be prepared, in the presence orabsence of a base, by carbonating micacocidine in an appropriatesolvent.

Carbonation can be performed in the same manner as the reaction inScheme 2 stated above. Out of the present compounds of the formula(I-4), the compound wherein R²¹ is CONR¹⁵ R¹⁶ and R²² is CONR¹⁹ R²⁰, canbe prepared, in the presence or absence of a base, by carbamoylingmicacocidin in an appropriate solvent. Carbamoylation can be performedin the same manner as the reaction in Scheme 2 stated above.

Out of the compounds of the formula (I-4) of the present invention, thecompound wherein R²¹ and R²² are different from each other, can beprepared by subjecting the compound of the formula (I-3) of the presentinvention which was obtained according to Scheme 3 stated above to thesame reaction as that in Scheme 2 stated above to introduce thesubstituent R²² which differs from R²¹.

The compound (I-4) of the invention thus obtained can be optionallypurified by conventional methods such as column chromatography andrecrystalization, or it is obtained by subjecting the compound (I-2)obtained by the reaction in Scheme 2 to the same reaction as that inScheme 3 and then subjecting the product to hydrolysis or catalyticreduction, if necessary. ##STR7## wherein R²³ presents optionallysubstituted alkyl, optionally substituted aralkyl, optionallysubstituted heteroaryl or optionally substituted heteroarylalkyl.

The compound of the the formula (I-5) of the invention can be preparedby alkylating or arylating micacocidine or the compound (I-1) of thepresent invention in an appropriate solvent in the presence or absenceof a base.

Alkylation or Arylation can be performed in the same manner as thereaction of Scheme 2 stated above, provided that the alkylating orarylating agent and a base are used in an amount of 1-3 equivalents,preferably 1-1.5 equivalents for micacocidin or the compound (I-1). Thecompound (I-5) can also be prepared by allowing the compound (I-2) toreact, in the presence of a dehydrating agent, for example,dicyclohexylcarbondiimide, with the corresponding alcohol (R²³ OH), orconverting the compound (I-2) to the reactive derivative such as acidchloride or activated ester, and then reacting the product with R²³ OH.

The compound (I-5) of the present invention thus obtained can optionallybe purified by conventional manner, for example, by columnchromatography and recrystalization. ##STR8## wherein R^(1') representsCONR⁵ R⁶ wherein R⁵ and R⁶ are as defined above, or CO--R⁷ --OR whereinR⁷ and R are as defined above.

The compound of the the formula (I-6) can be prepared, in the presenceor absence of a base, through amidation of micacocidin or the compound(I-1) of the present invention by condensing them with an amine using anappropriate condensing agent in an appropriate solvent.

The amines include for example α-amino acid ester such as L-alaninemethyl ester, L-seline methyl ester, L-cystine methyl ester; alkylamines such as methylamine, ethanolamine, dimethylamine; hydroxylaminessuch as hydroxylamine, N-methylhydroxylamine, methoxylamine; andhydrochloride thereof. They are used in an amount of 1-5 equivalents,preferably 1-2 equivalents of micacocidin or the compound (I-1).

Solvents to be used include carbohydrate halides such asdichloromethane, chloroform, chlorobenzene and the like; ethers such asdiethyl ether, tetrahydrofurane, dioxane and the like; amides such asN,N-dimethylformamide, N,N-dimethylacetoamide; sulfoxides such asdimethyl sulfoxide; carbohydrates such as hexane, benzene, toluene;ketones such as acetone, ethylmethyl ketone; nitrites; and a mixturethereof.

Bases to be used include amines such as triethylamine,N,N-diisopropylethylamine. They are used in an amount of 1-5equivalents, preferably 1-3 equivalents of micacocidin or the compound(I-1).

Condensing agents to be used include carbodiimides such as1,3-dicyclohexylcarbodiimide (DCC), diethyl cyanophosphate (DEPC),diphenyl phosphorylazide (DPPA),benzotriasol-1-yloxytris(dimethylamino)phosphonium-hexafluorophosphate(Bop reagent), bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl).They are used in an amount of 1-5 equivalents, preferably 1-2equivalents of micacocidin or the compound (I-1).

The reaction temperature is -20 to 80° C., preferably 0 to 40° C., andthe reaction time is 5 minutes to 24 hours, preferably 30 minutes to 15hours.

The compound (I-6) thus obtained can optionally be purified byconventional methods such as column chromatography, thin layerchromatography, and recrystalization. ##STR9## wherein R⁸ and R²³ are asdefined above.

The compound of the formula (I-7) of the present invention can beprepared by reducing the compound (I-1) or (I-5) in an appropriatesolvent.

The reducing agents include boron compounds, such as diborane, andlithium boron hydride; aluminum compounds such as lithium aluminumhydride, and diisobutyl aluminum hydride. The reducing agents are usedin an amount of 1-5 equivalents, preferably 1-3 equivalents of compound(I-1) or the compound (I-5).

Solvents to be used include ethers such as diethyl ether,tetrahydrofuran, dioxane; carbohydrates such as hexane, benzene, andtoluene.

The reaction temperature is -78 to 60° C., preferably -78 to 40° C., andthe reaction time is 10 minutes to 24 hours, preferably 30 minutes to 15hours.

The compound (I-7) thus obtained can optionally be purified byconventional methods such as column chromatography, thin layerchromatography, and recrystalization. ##STR10## wherein R²³ and R²⁵ arethe same or different and represent optionally substituted alkyl,optionally substituted aralkyl, optionally substituted heteroaryl oroptionally substituted heteroarylalkyl.

Out of the compounds of the formula (I-8), the compound wherein R²³ andR²⁵ are the same, can be prepared, in the presence or absence of a base,by alkylating or arylating micacocidin in an appropriate solvent.Alkylation or arylation can be accomplished in the same manner as thereaction of Scheme 2 mentioned above. Out of the compounds of theformula (I-8), the compound wherein R²³ and R²⁵ differ from each other,can be prepared, for example, by subjecting the compound of the formula(I-2) to the same reaction as that in Scheme 3 mentioned above tointroduce R²³ which differs from R²² of the formula (I-2).

The compound (I-8) thus obtained can optionally be purified by inconventional manner, for example, by column chromatography, thin layerchromatography, or recrystalization. ##STR11## wherein R²³ and R²⁴ arethe same or different and represent optionally substituted alkyl,optionally substituted aralkyl, optionally substituted heteroaryl oroptionally substituted heteroarylalkyl.

Out of the compounds of the formula (I-9), the compound wherein R andR24 are the same, can be prepared, in the presence or absence of a base,by alkylating or arylating micacocidine or the compound (I-1) in anappropriate solvent.

Alkylation or arylation can be accomplished in the same manner as thereaction of Scheme 2 stated above. Out of the compounds of the formula(I-9), the compound wherein R²³ and R²⁴ are different from each other,can be prepared, for example, by subjecting the compound of the formula(I-5) to the same reaction as that in Scheme 3 stated above to introduceR²⁴ which differs from R²³ of the formula (I-5).

The compound (I-9) thus obtained can optionally be purified inconventional manner, for example, by column chromatography, thin layerchromatography, are recrystalization. ##STR12## wherein R²³, R²⁴ and R²⁵are as defined above.

Out of the compounds of the formula (I-10), the compound wherein R²³,R²⁴ and R²⁵ are identical, can be prepared, in the presence or absenceof a base, by alkylating or arylating micacocidine in an appropriatesolvent.

Alkylation or arylation can be accomplished in the same manner as thereaction of Scheme 2 stated above.

Out of the compounds of the formula (I-10), the compound wherein any oneof R²³ and R²⁴ and R²⁵ is different from others, can be prepared, forexample, by subjecting the compound of the formula (I-4), (I-8) or (I-9)to the same reaction as that in Scheme 5, 3 or 2 stated above,respectively, to introduce a different substituent.

The compound (I-10) thus obtained can optionally be purified byconventional methods such as column chromatography, thin layerchromatography, and recrystalization. ##STR13## wherein R¹ and R² are asdefined above.

The compound of the formula (I-12) can be prepared by oxidizing thecompound of the formula (I-11).

The oxidizing agents include dimethyl sulfoxide (DMSO) used in thepresence of an appropriate electrophilic reagent in the presence orabsence of a base. They are used in an amount of 1-5 equivalents,preferably 1-3 equivalents of the compound of the formula (I-11).

Bases to be used with DMSO as an oxidizing agent include amines such astriethylamine, N,N-diisopropylethylamine. They are used in an amount of1-5 equivalents, preferably 1-3 equivalents of the compound of theformula (I-11).

The electrophilic reagents to be used with DMSO as an oxidizing agentinclude, for example, carbodiimides such as 1,3-dicyclohexylcarbodiimide(DCC); acid anhydrides such as acetic anhydride; acid chlorides such asoxalyl chloride; pyridine-sulfur trichloride complex. They are used inan amount of 1-5 equivalents, preferably 1-3 equivalents of the compoundof the formula (I-11).

The reaction temperature is -78 to 60° C., preferably -78 to 40° C., andthe reaction time is 10 minutes to 24 hours, preferably 30 minutes to 15hours.

The compound of the present invention (I-12) thus obtained canoptionally be purified by a conventional method, for example, columnchromatography, thin layer chromatography, recrystalization, etc. Thecompound of the present invention represented by the formula (I-11)which is used as a starting material in this reaction can be prepared bycombining the reactions mentioned above.

The compound of the present invention may form a salt through itscarboxylic acid moiety. Such salts are alkali metal salts, such assodium salt, potassium salt and lithium salt; ammonium salt; and organicsalts, such as methyl ammonium salt, dimethyl ammonium salt, triethylammonium salt, tetrabuthyl ammonium salt and the like.

The compound of the present invention represented by the formula (I) canbe reacted with an aqueous solution of a bivalent or trivalent metalsalt, such as chloride, sulfate or nitrate, to obtain a metal complex.

Bivalent or trivalent metals include Zn²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺,Mn²⁺, Mn³⁺, Co²⁺, Mg²⁺, Ca²⁺, Sr ²⁺, Ba²⁺, V²⁺, V³⁺, Cr²⁺, Al³⁺, Sn²⁺,Pb²⁺, Ag²⁺ or the like.

The present invention further provides a pharmaceutical composition or aveterinary medicine containing the compound represented by the formula(I) of the present invention in combination with one or morepharmaceutical acceptable carriers.

The compound represented by the formula (I) of the present inventionhas, among others, anti-mycoplasma activity, antibacterial activity,antifungal activity and immunosuppressive activity, and therefore, canbe used as an anti-mycoplasma agent, antibacterial agent, antifungalagent and immunosuppressor.

When used as a medicament, the compound of the invention can be orallyor parenterally administered. For oral administration, the compound ofthe present invention can be formulated into conventional formulations,such as a solid formulation such as tablets, powders, granules andcapsules; aqueous solutions; oily suspensions; or liquid preparationssuch as syrups or elixirs. For parenteral administration, it can beformulated into aqueous or oily suspensions, or suppositories. Each ofthem can be produced using carriers such as conventional exipients,binders, aqueous and oily solvents, emulsions and suspensions, ifnecessary, and optionally contains other additives such as preservativesor stabilizing agents.

The compound of the present invention is usually administered at a doseof 100-2000 mg per day, preferebly 500-100 mg per day, which may bedivided into 1 to 5 portions, although the dose will vary depending onthe administration route, ages, weights and the condition of patients,and the type of diseases.

When used as a veterinary medicine, it can be orally or parenterallyadministered to domestic fowls and animals such as chickens, swine andcattle. Generally, for oral administration, it is administered incombination with a commonly used carrier (such as defatted rice bran,defatted soy bean meal, wheat bran, kaolin, talc, calcium carbonate,lactic acid and water). However, it is preferable that such combinationor the compound of the present invention is administered after combinedwith feed for animals or water. The feed for animals may be the oneswhich are commonly used as feed for animals, such as corn, wheat bran,rice, wheat, cotton seed meal, milo, soy bean meal, fish meal, defattedrice bran, oils and fats, alfalfa, calcium carbonate, calcium phosphate,sodium chloride, choline chloride, vitamins such as vitamin A, vitaminD, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B12, calciumpantothenate, nicotinic acid amide and folic acid, and inorganic saltssuch as magnesium sulfate, ferric sulfate, cupric sulfate, zinc sulfate,potassium iodide and cobalt sulfate. Part or all of them may be combinedto use.

The contents of the compound of the present invention in feed are in therange of 100-2000 ppm, and preferably 500-1000 ppm.

For parenteral administration, it can be used in the same manner as thatof the aforementioned medicament.

The compound of the present invention is generally administered at adose of 50-2000 mg, preferably 100-500 mg for oral administration, and50-100 mg, preferably 10-50 mg for parenteral administration, per kg ofbody weights of animal per day. The compound is administered for severalconsecutive days.

EXAMPLES

The present invention will be described in detail by means of, but notlimited to, the following Reference Examples and Working Examples.

REFERENCES Preparation of Mycacocidin by Culturing Pseudomonas sp. No.57-250

1) Seed Culture

Bennet medium (1% glucose, 0.2% yeast extract, 0.1% meat extract and0.2% casamino acid, pH 7) was autocraved at 121° C. for 20 min. andPseudomonas sp. No.57-250 was inoculated and cultured at 28° C. using arotary shaker at 140 rpm for 18 hours.

2) Main Culture

Eighty L of fermentation medium (1% glucose, 2% dextrin, 1% yeastextract, 1% Pharmamedia, 0.0002% cupric sulfate hexahydrate, 0.01% zincsulfate heptahydrate, pH 6.5) was added to jars (2×50 L, 2×30 L)followed by adding anti-foaming agent (Polypropylene glycol 2000) at afinal concentration of 0.01%. After sterilizing at 121° C. for 20 min.and cooling, the seed culture was inoculated at a final concentration of1%. Then, they were cultured under the following conditions: at 28° C.,agitating at 290-600 rpm, aerating at 25 L/min. for 50-L jar and 15L/min. for 25-L jar, cultured for 42 hours.

3) Extraction and Isolation

(1) To 8.5 L of culture (pH 8.3), the equal amount of ethyl acetate wasadded and then liquid-liquid preparative extraction was performed twice.760 mg of the culture was fractioned by column chromatography (140 g ofsilica gel, 70-230 mesh, Merck No.7734). Micacocidin was eluted withdichloromethane/methanol (95:5, v/v) solvent to give 11 mg of crudeelute, and then, the eluate was partitioned to purify using thin layerchromatography (silica gel plate, 20×20 cm, 0.5 mm, Merck No.5744).Thus, after said eluate was developed 15-cm with chloroform/methanol(9:1, v/v) solvent, a bright blue band (irradiation with a UV lamp at365 nm) around Rf value 0.5 was collected and extracted with a mixtureof chloroform/methanol (8:2, v/v) to elute micacocidin. Furtherpurification was conducted with a Sephadex LH-20 column (ID 15 mm×870mm). Thus, the sample was dissolved in a small amount of methanol,followed by isolation with methanol. A fraction (6.6 mg) containingmicacocidin was recrystallized from methanol/ethyl acetate to give 3.6mg of colourless and rhombic board-shaped crystals. m.p.: 226-228° C.(Decomposition).

(2) About 70 L of the culture was centrifuged and separated into 66 L ofthe supernatant and the bacterial cells. 3.3 kg of HP-20 was added tothe supernatant and agitated at room temperature for 1 hour. HP-20 wasfiltrated by a mesh filter, washed with water and then filled in acolumn. After washing with 40 L of deionized water, the elution withmethanol gives 12 L of the active fraction. The fraction wasconcentrated in vacuo and then eluted with ethyl acetate. On the otherhand, the bacterial cells were extracted with methanol, which wasfiltered, concentrated in vacuo and the residue was extracted with ethylacetate. The ethyl acetate extracts obtained above from the supernatantand the bacterial cells were combined and concentrated to dryness,resulting in 11.3 g of a crude extract.

The crude extract was dissolved in chloroform, charged in a silica gelcolumn (350 g of silica gel, column: 50×350 mm) and then developed withchloroform-methanol. After washing with 500 mL of chloroform and 1.6 Lof chloroform-methanol (98:2), the column was eluted with 1.3 L ofchloroform-methanol (9:1) to give 5.7 g of a crude fraction containingcompound B. Repeated recrystallization of the crude fraction from ethylacetate-methanol provided 1.73 g of compound B (micacocidin) ascrystals.

Example 1 ##STR14##

Two mg of micacocidin (hereinafter referred to as Compound 1) wasdissolved in 2 mL of dichloromethane, followed by shaking with 4 mL of1N hydrochloric acid (or 5% potassium hydrogen sulfate) with stirring.The dichloromethane layer was washed and dried in vacuo to give 1.8 mgof compound 2.

Molecular formula: C₂₇ H₃₉ N₃ O₄ S₃ ; SIMS m/z=566[M+H]⁺ ; ¹H-NMR(CDCl₃):0.90 ppm(3H, t, J=7.1 Hz), 1.31 ppm(3H, s), 1.32 ppm(3H,s), 1.32-1.38 ppm(4H, m), 1.59 ppm(3H, s), 1.59-1.63 ppm(2H, m), 2.63ppm(3H, s), 2.86-3.02 ppm(3H, m), 3.17 ppm (1H, dd, J=11.2 Hz, 7.8 Hz),3.18 ppm(1H, d, J=11.5 Hz), 3.26 ppm (1H dd, J=11.2 Hz, 7.1 Hz), 3.34ppm(1H, m), 3.47 ppm(1H, dd, J=11.2 Hz, 8.8 Hz), 3.61 ppm(1H, d, J=6.8Hz), 3.65 ppm(1H, d, J=11.5 Hz), 4.21 ppm(1H, d, J=8.7 Hz), 4.76 ppm(1H,ddd, J=8.8 Hz, 8.7 Hz, 7.8 Hz), 6.71 ppm(1H, dd, J=7.8 Hz, 1.2 Hz), 6.85ppm(1H, dd, J=8.3 Hz, 1.2 Hz), 7.21 ppm(1H, dd, J=8.3 Hz, 7.8 Hz). ¹³C-NMR(CDCl₁):14.05 ppm(q), 22.50 ppm(t), 23.89 ppm(q), 24.15 ppm(q),24.20 ppm(q), 31.89 ppm(t), 32.17 ppm(t), 35.03 ppm(t), 35.40 ppm (t),36.73 ppm(t), 41.12 ppm(t), 45.61 ppm(s), 46.12 ppm(q), 72.72 ppm(d),78.04 ppm(d), 78.08 ppm(d), 79.35 ppm(d), 83.87 ppm (s), 115.43 ppm(d),116.29 ppm(s), 121.46 ppm(d), 132.25 ppm(d), 143.80 ppm(s), 159.57ppm(s), 172.11 ppm(s), 175.08 ppm(s), 181.87 ppm(s).

Example 2 ##STR15##

Eighty eight mg (0.15 mM) of compound 2 was dissolved in 1.0 mL ofmethanol. To the solution were added 29.7 mg (0.23 mM) ofN-diisopropylethylamine at room temperature, and then, 0.12 mL (0.24 mM)of a 2M trimethylsilyldiazomethane solution with stirring at roomtemperature, followed by stirring over night. After completion of thereaction, 3 mL of methanol was added. Subsequently, 2 mL of 1N sodiumhydroxide were added under ice-cooling, followed by stirring for onehour at room temperature. The reaction was adjusted to pH 6 with 2Nhydrochloric acid under ice-cooling, extracted 3 times withdichloromethane, washed 2 times with water, and then, dried withanhydrous sodium sulfate. Then, the solvent was evaporated to obtain 65mg of crude product. Isolating and purifying the product using PHLCgives the purposed compound 7.

Molecular formula: C₂₈ H₄₁ N₃ O₄ S₃ ; FAB MS: m/z=580[M+H]⁺ ; ¹H-NMR(CDCl₃): 0.89 ppm(3H, t, J=7.1 Hz), 1.27 ppm(3H, s), 1.30-1.34ppm(4H, m), 1.36 ppm(3H, s), 1.39 ppm(3H, s), 1.57-1.65 ppm(2H, m), 2.54ppm(3H, s), 2.59-2.63 ppm(2H, m), 2.99 ppm(1H, dd, J=11.5 Hz, 5.1 Hz),3.03 ppm(1H, d, J=11.7 Hz), 3.24 ppm(1H, dd, J=11.5 Hz, 7.3 Hz), 3.27ppm(1H, dd, J=11.2 Hz, 9.0 Hz), 3.32 ppm(1H, dt, J=7.3 Hz, 5.1 Hz), 3.50ppm(1H, d, J=5.1 Hz), 3.57 ppm(1H, dd, J=11.2 Hz, 8.5 Hz), 3.73 ppm(1H,d, J=11.7 Hz), 3.81 ppm(3H, s), 4.30 ppm(1H, d, J=9.3 Hz), 4.91 ppm(1H,ddd, J=9.3 Hz, 9.0 Hz, 8.5 Hz), 6.73 ppm(1H, d, J=7.8 Hz), 6.82 ppm(1H,d, J=7.3 Hz), 7.25 ppm(1H, dd, J=7.8 Hz, 7.3 Hz). ¹³ C-NMR(CDCl₃):14.03ppm(q), 22.53 ppm(t), 23.92 ppm(q), 24.57 ppm (q), 26.21 ppm(q), 30.79ppm(t), 31.81 ppm(t), 32.72 ppm(t), 37.04 ppm (t), 37.21 ppm(t), 40.79ppm(t), 45.24 ppm(s), 45.75 ppm(q), 55.95 ppm (d), 71.97 ppm(d), 78.93ppm(d), 80.55 ppm(d), 81.25 ppm(d), 84.26 ppm (s), 108.45 ppm(d), 121.38ppm(d), 122.54 ppm(s), 130.16 ppm(d), 142.55 ppm(s), 156.94 ppm(s),168.16 ppm(s), 174.57 ppm(s), 181.33 ppm (s).

Example 3 ##STR16##

8.9 mg (94.9% contained) of compound 2 was dissolved in 1 mL ofmethanol, to which 15.58 μL of 1N sodium hydroxide was added andstirred. After few minutes, the mixture was evaporated to dryness invacuo, and the residue was dissolved in 0.5 ml of water and lyophilizedto give 9 mg of compound 9.

Molecular formula: C₂₇ H₃₈ N₃ O₄ S₃ Na

Example 4 ##STR17##

150 mg (0.265 mM) of compound 2 was dissolved in 2 mL of methanol, towhich 0.74 ml (0.37 mM) of a 2 M trimethylsilyldiazomethane/hexanesolution was added at room temperature with stirring. The mixture waskept standing for 50 minutes at room temperature and concentrated, andthen, purified by PTLC to give 88 mg of oily compound 12. A sample foridentification of the structure and assay was further purified by HPLC.

Molecular formula: C₂₈ H₄₁ N₃ O₄ S₃ ; SIMS: m/z=580[M+H]⁺ ; ¹H-NMR(CDCl₃): 0.88 ppm(3H, t, J=7.1 Hz), 1.29-1.37 ppm(4H, m), 1.30ppm(3H, s), 1.33 ppm(3H, s), 1.51 ppm(3H, s), 1.58-1.62 ppm(2H, m), 2.62ppm(3H, s), 2.83-3.04 ppm(2H, m), 2.91 ppm(1H, dd, J=11.7 Hz, 4.6 Hz),3.10 ppm(1H, d, J=11.4 Hz), 3.14 ppm(1H, dd, J=11.4 Hz, 7.8 Hz), 3.23ppm(1H, dd, J=11.7 Hz, 7.1 Hz), 3.40 ppm(1H, ddd, J=7.1 Hz, 6.6 Hz, 4.6Hz), 3.45 ppm(1H, dd, J=11.4 Hz, 8.8 Hz), 3.51 ppm(1H, d, J=6.6 Hz),3.61 ppm(1H, d, J=11.4 Hz), 3.77 ppm(3H, s), 4.18 ppm(1H, d, J=9.2 Hz),4.35 ppm(1H, br), 4.76 ppm(1H, ddd, J=9.2 Hz, 8.8 Hz, 7.8 Hz, 6.70ppm(1H, dd, J=7.6 Hz, 1.2 Hz) 6.85 ppm(1H, dd, J=8.3 Hz, 1.2 Hz), 7.20ppm(1H, dd, J=8.3 Hz, 7.6 Hz), 12.86 ppm(1H, br). ¹³ C-NMR(CDCl₃):14.04ppm(q), 21.90 ppm(q), 22.48 ppm(t), 23.41 ppm (q), 26.07 ppm(q), 31.87ppm(t), 32.18 ppm(t), 35.02 ppm(t), 35.42 ppm (t), 36.65 ppm(t), 41.25ppm(t), 45.65 ppm(s), 45.89 ppm(q), 52.73 ppm (q), 73.02 ppm(d), 77.73ppm(d), 78.06 ppm(d), 79.67 ppm(d), 83.69 ppm (s), 115.36 ppm(d), 116.25ppm(s), 121.36 ppm(d), 132.15 ppm(d), 143.77 ppm(s), 159.69 ppm(s),171.73 ppm(s), 173.72 ppm(s), 178.05 ppm (s).

Example 5 ##STR18##

To a suspension consisting of 10.7 mg of compound 12, 50 mg of potassiumcarbonate and 190 μl of dimethylformamide, 5.0 mg of4-chlorobenzylbromide was added at room temperature and then stirred for2 hours. To the reaction was added dichloromethane, and the mixture waswashed with an excessive amount of 5% potassium hydrogen sulfatesolution. The organic layer was dried with anhydrous sodium sulfate, andthen, the solvent was removed by evaporation in vacuo, and the resultantresidue was subjected to silica gel column chlomatography (elutedconsecutively with 20% ethyl acetate/hexane, 30% ethyl acetate/hexane)togive 10.7 mg of compound 11.

Molecular formula: C₃₅ H₄₆ CIN₃ O₄ S₃ ; ¹ H-NMR(CDCl₃): 0.89 (3H, t,J=7.1 Hz), 1.27 ppm(3H, s) 1.31 ppm(3H, s), 1.32-1.34 ppm(4H, m), 1.50ppm(3H, s), 1.58-1.62 ppm(2H, m), 2.47 ppm(3H, s), 2.60-2.64 ppm(2H, m),2.81 ppm(1H, dd, J=11.5 Hz, 3.7 Hz), 3.09 ppm(1H, d, J=11.2 Hz), 3.16ppm(1H, dd, J=11.5 Hz, 7.1 Hz), 3.30 ppm(1H, ddd, J=7.1 Hz, 6.6 Hz, 3.7Hz), 3.34 ppm(1H, dd, J=11.3 Hz, 7.1 Hz), 3.45 ppm(1H, d, J=6.6 Hz),3.54 ppm(1H, dd, J=11.3 Hz, 8.8 Hz), 3.60 ppm(1H, d, J=11.2 Hz), 3.77ppm(3H, s), 4.27 ppm(1H, d, J=8.8 Hz), 4.71 ppm(1H, br), 4.82 ppm(1H,dt, J=7.1 Hz, 8.8 Hz), 5.04 ppm(2H, s), 6.72 ppm(1H, d, J=7.6 Hz), 6.85ppm(1H, d, J=7.1 Hz), 7.21 ppm(1H, dd, J=7.6 Hz, 7.1 Hz), 7.33 ppm(2H,d, J=8.5 Hz), 7.38 ppm(2H, d, J=8.5 Hz). ¹³ C-NMR(CDCl₃): 14.04 ppm(q),22.08 ppm(q), 22.54 ppm(t), 23.40 ppm ppm(q), 26.10 ppm(q), 30.88ppm(t), 31.83 ppm(t), 32.85 ppm(t), 36.37 ppm ppm(t), 37.02 ppm(t),41.26 ppm(t), 44.86 ppm(q), 45.67 ppm(s), 52.73 ppm ppm(q), 69.58ppm(t), 72.25 ppm(d), 77.91 ppm(d), 79.49 ppm(d), 81.71 ppm (d), 83.61ppm(s), 109.75 ppm(d), 121.99 ppm(d), 123.41 ppm(s), 128.49 ppm(d),128.61 ppm(d), 129.95 ppm(d), 133.46 ppm(s), 135.60 ppm ppm(s), 142.79ppm(s), 155.64 ppm(s), 166.41 ppm(s), 173.80 ppm(s), 178.00 ppm(s).

Example 6 ##STR19##

To 12 mg of compound 2 dissolved in 0.4 ml of methanol, a solution ofdiazomethane in ether was added and allowed to react at room temperaturefor 21 hours. After completion of the reaction, the mixture wasconcentrated to dryness in vacuo. Preparative TLC gave 12 mg of compound14, which was further purified by preparative HPLC.

Molecular formula: C₂₉ H₄₃ N₃ O₄ S₃ ; FAB MS:m/z=616[M+Na]⁺, 594{M+H]⁺ ;¹ H-NMR(CDCl₃): 0.88 ppm(3H, t, J=7.1 Hz), 1.28 ppm(3H, s), 1.32 ppm(3H,s), 1.28-1.33 ppm(4H, m), 1.51 ppm(3H, s), 1.59-1.63 ppm(2H, m),2.58-2.65 ppm(2H, m), 2.61 ppm(3H, s), 2.83 ppm(1H, dd, J=11.5 Hz, 3.6Hz), 3.09 ppm(1H, d, J=11.2 Hz), 3.20 ppm(1H, dd, J=11.5 Hz, 7.1 Hz),3.32 ppm(1H, m), 3.37 ppm(1H, dd, J=11.2 Hz, 7.3 Hz), 3.47 ppm(1H, d,J=6.6 Hz), 3.55 ppm(1H, dd, J=11.2 Hz, 8.7 Hz), 3.60 ppm(1H, d, J=11.2Hz), 3.78 ppm(3H, s), 3.79 ppm(3H, s), 4.38 ppm(1H, d, J=8.3 Hz), 4.74ppm(1H, br), 4.87 ppm(1H, ddd, J=8.7 Hz, 8.3 Hz, 7.3 Hz), 6.73 ppm(1H,d, J=8.3 Hz), 6.83 ppm(1H, d, J=7.6 Hz), 7.24 ppm(1H, dd, J=8.3 Hz, 7.6Hz). ¹³ C-NMR(CDCl₃): 13.99 ppm(q), 22.04 ppm(q), 22.50 ppm(t), 23.35ppm (q), 26.04 ppm(q), 30.85 ppm(t), 31.78 ppm(t), 32.77 ppm(t), 36.30ppm (t), 36.73 ppm(t), 41.21 ppm(t), 44.76 ppm(q), 45.61 ppm(s), 52.66ppm (q), 55.80 ppm(q), 72.16 ppm(d), 77.86 ppm(d), 79.44 ppm(d), 81.56ppm (d), 83.59 ppm(s), 108.35 ppm(d), 121.44 ppm(d), 122.75 ppm(s),129.93 ppm(d), 142.55 ppm(s), 156.89 ppm(s), 166.38 ppm(s), 173.73 ppm(s), 177.98 ppm(s).

Example 7 ##STR20##

To 24.0 mg of compound 14 dissolved in 0.4 mL of 1, 2-dichloroethane, 8mg of 4-pyrolydinopyridine and 10 μl ofR-(-)-α-methoxy-α-(trifluoromethyl) phenylacethyl chloride(R-(-)-MTPAC1) were added and stirred at 55° C. After 30 minutes, 70μlof N,N-diisopropylethylamine and 10 μl ofR-(-)-α-methoxy-α-(trifluoromethyl) phenylacethylchloride were added tothe mixture, and stirred for 30 minutes. Ten μl ofR-(-)-α-methoxy-α-(trifluoromethyl)phenylacethyl chloride wassupplemented and stirred for 30 minutes. The reaction was furtherstirred at 70° C. for 1 hour and allowed to cool. The reaction wasdiluted with dichloromethane and then washed with an aqueous solution of5% potassium hydrogen sulfate. The organic layer was dried withanhydrous sodium sulfate and the solvent was removed by evaporation invacuo. The residue was dissolved in 1.0 ml of diethyl ether and 0.5 mlof methanol, and then, an exessive amount oftrimethylsilyldiazomethane/hexane solution was added thereto and allowedto react. The reaction mixture was diluted with ethyl acetate and washedconsecutively with an aqueous solution of 5% potassium hydrogen sulfateand saturated saline. The organic layer was dried with anhydrous sodiumsulfate, and the solvent was removed in vacuo. The residue was subjectedto silica gel column chromatography (eluted consecutively with 20% ethylacetate/hexane and 50% ethyl acetate/hexane), and the aimed compound wasfurther purified using HPLC to give 9.3 mg of compound 15.

Molecular formula: C₃₉ H₅₀ F₃ N₃ O₆ S₃ ; ¹ H-NMR(CDCl₃ : 0.86 ppm(3H, t,J=6.8 Hz), 1.19 ppm(3H, s), 1.24-1.30 (4H, m), 1.31 ppm(3H, s), 1.51ppm(3H, s), 1.51-1.60 ppm(2H, m), 2.42 ppm(3H, s), 2.57-2.62 ppm(2H, m),3.01 ppm(1H, dd, J=11.2 Hz, 6.3 Hz), 3.10 ppm(1H, dd, J=11.2 Hz, 7.8Hz), 3.14 ppm(1H, d, J=11.4 Hz), 3.25 ppm(1H, ddd, J=7.8 Hz, 7.5 Hz, 6.3Hz), 3.41 ppm (1H, dd, J=11.0 Hz, 9.1 Hz), 3.49 ppm(1H, dd, J=11.0 Hz,9.3 Hz), 3.53 ppm(3H, s), 3.69 ppm(1H, d, J=11.4 Hz), 3.76 ppm(3H, s),3.78 ppm(3H, s), 4.57 ppm(1H, d, J=5.9 Hz), 4.94 ppm(1H, ddd, J=9.3 Hz,9.1 Hz, 5.9 Hz), 5.64 ppm(1H, d, J=7.5 Hz), 6.72 ppm(1H, d, J=7.8 Hz),6.81 ppm(1H, d, J=7.1 Hz), 7.20-7.35 ppm(3H, m), 7.24 ppm (1H, dd, J=7.8Hz, 7.1 Hz), 7.65 ppm(2H d, J=7.8 Hz) ¹³ C-NMR(CDCl₃): 14.02 ppm(q),21.05 ppm(q), 22.50 ppm(t), 23.25 ppm(q), 26.48 ppm(q), 31.04 ppm(t),31.38 ppm(t), 32.88 ppm(t), 33.49 ppm(t), 35.94 ppm(t), 40.59 ppm(q),41.70 ppm(t), 44.92 ppm(s), 52.85 ppm(d), 55.30 ppm(d), 55.93 ppm(d),71.29 ppm(d), 78.37 ppm(d), 80.50 ppm(d), 80.77 ppm(d), 84.01 ppm(s),84.92 ppm(q), 108.38 ppm(d), 121.51 ppm(d), 123.07 ppm(s), 123.45ppm(q), 128.09 ppm(d), 128.40 ppm (d), 129.47 ppm(d), 129.82 ppm(d),131.72 ppm(s), 142.76 ppm(s), 156.93 ppm(s), 165.97 ppm(s), 166.31ppm(s), 173.37 ppm(s), 176.82 ppm (s).

Example 8 ##STR21##

7.0 μl of oxalyl chloride was dropped into a solution of 9.0 μl ofdimethylsulfoxide in 0.4 ml of dichloromethane, while cooling with dryice/acetone, with stirring. Then, a solution consisting of 23.1 mg ofcompound 14 and 0.4 ml of dichloromethane was dropped into the mixture,which was stirred for 30 minutes. Then, triethylamine was dropped intothe solution. The temperature was gradually allowed to raise up to roomtemperature and the mixture was stirred for 30 minutes. Then, thereaction mixture was diluted with dichloromethane and washed with anaqueous solution of 5% potassium hydrogen sulfate. The organic layer wasdried with anhydrous sodium sulfate and the solvent was removed byevaporation in vacuo. The residue was subjected to silica gel columnchromatography (eluted consecutively with 30% ethyl acetate/hexane and50% ethyl acetate/hexane, ethyl acetate of 10% methanol/ethyl acetate),which was repurified with silica gel column chromatography to give 4.0mg of compound 16.

Molecular formula: C₂₉ H₄₁ N₃ O₄ S₃ ; ¹ H-NMR(CDCl₃): 0.88 ppm(3H, t,J=7.1 Hz), 1.32-1.37 ppm(4H, m), 1.46 ppm(3H, s), 1.51 ppm(3H, s),1.54-1.62 ppm(2H, m), 1.56 ppm(3H, s), 2.40 ppm(3H, s), 2.62-2.67ppm(2H, m), 2.81 ppm(1H, t, J=10.2 Hz), 3.06 ppm(1H, dd, J=10.2 Hz, 5.4Hz), 3.18 ppm(1H, d, J=11.2 Hz), 3.49 ppm(1H, dd, J=11.0 Hz, 9.3 Hz),3.74 ppm(1H, dd, J=11.0 Hz, 9.3 Hz), 3.75 ppm(1H, d, J=11.2 Hz), 3.80ppm(3H, s), 3.81 ppm(3H, s), 4.11 ppm(1H, dd, J=10.2 Hz, 5.4 Hz), 4.66ppm(1H, d, J=3.9 Hz), 5.16 ppm(1H, dt, J=3.9 Hz, 9.3 Hz), 6.72 ppm(1H,dd, J=8.3 Hz, 0.7 Hz), 6.82 ppm(1H, dd, J=7.8 Hz, 0.7 Hz), 7.23 ppm(1H,dd, J=8.3 Hz, 7.8 Hz). ¹³ C-NMR(CDCl₃): 14.04 ppm, 22.56 ppm, 23.66 ppm,23.93 ppm, 24.40 ppm, 31.14 ppm, 31.82 ppm, 32.85 ppm, 34.11 ppm, 34.54ppm, 39.17 ppm, 42.00 ppm, 35.03 ppm, 54.27 ppm, 55.95 ppm, 73.36 ppm,74.89 ppm, 79.79 ppm, 84.44 ppm, 108.31 ppm, 121.45 ppm, 122.82 ppm,129.88 ppm, 142.75 ppm, 156.88 ppm, 167.15 ppm, 173.18 ppm, 174.21 ppm,206.17 ppm.

Example 9 ##STR22##

34.9 mg(77.7% contained)of compound 2 was dissolved in 1.0 ml ofdimethylformamide, to which, while stirring under ice-cooling, 11 mg ofN-methylhydroxylamine hydrochloride, 54 mg of BOP reagent(benzotriazol-1-yloxytrisppm(dimethylamino) phosphoniumhexafluorophosphate) and 17 mg of N,N-diisopropylethylamine/10.0 ml ofbenzene were added, stirred for 15 minutes, and then, the mixture isallowed to react for 16 hours at room temperature. Water was added tothe reaction, which was eluted with ethyl acetate. The solution wasdried with anhydrous sodium sulfate, and dried in vacuo to give 58 mg ofcompound 24. The residue was purified by preparative TLC.

Molecular formula:C₂₈ H₄₀ N₄ O₄ S₃ Zn; SIMS:m/z=657[M+H]⁺, 441; ¹H-NMR(CDCl₃):0.91 ppm(3H, t, J=7.0 Hz), 1.34 ppm(3H, s), 1.34-1.37ppm(4H, m), 1.54-1.57 ppm(2H, m), 1.57 ppm(3H, s), 1.64 ppm(3H, s), 2.30ppm(1H, m), 2.31 ppm(3H, s), 2.78 ppm(1H, dd, J=12.5 Hz, 7.1 Hz), 2.92ppm(3H, s), 3.01-3.05 ppm(3H, m), 3.19ppm (1H, m), 3.57 ppm(1H, m), 3.58ppm(1H, d, J=12.6 Hz), 3.59 ppm(1H, m), 3.74 ppm(1H, d, J=12.6 Hz), 3.94ppm(1H, d, J=10.8 Hz), 4.44 ppm (1H, dt, J=10.8 Hz, 9.8 Hz), 6.61ppm(1H, d, J=7.0 Hz), 6.62 ppm(1H, d, J=7.0 Hz), 7.16 ppm(1H, t, J=7.0Hz), 11.68 ppm(1H, br). ¹³ C-NMR (CDCl₃):14.18 ppm, 20.75 ppm, 22.55ppm, 25.38 ppm, 29.41 ppm, 31.52 ppm, 33.19 ppm, 35.56 ppm, 35.74 ppm,37.31 ppm, 37.75 ppm, 38.24 ppm, 45.28 ppm, 47.83 ppm, 72.16 ppm, 74.86ppm, 77.59 ppm, 81.13 ppm, 83.45 ppm, 119.25 ppm, 120.68 ppm, 121.26ppm, 133.50 ppm, 145.80 ppm, 165.95 ppm, 171.59 ppm, 173.64 ppm, 187.38ppm.

Example 10 ##STR23##

21 mg (0.03 mM) of compound 2 was dissolved in 1.0 ml ofdimethylformamide, to which, while stirring under ice-cooling, 13 mg(0.16 mM) of N-methylhydroxylamine hydrochloride and 57 mg (0.13 mM) ofBOP reagent were added. After stirring for 10 minutes at the sametemperature, a solution of 21 mg (0.16 mM) of N,N-diisopropylethylaminein 1 ml of benzene was added to the reaction, and the mixture wasstirred for 15 minutes, and then, stirred for 2 days at roomtemperature. To the reaction was added water, and the mixture wasextracted with ethyl acetate, washed with water, dried with anhydroussodium sulfate, and the solvent was removed by evaporation to obtain 36mg of a partially purified product of compound 25. Isolation andpurification by using HPLC gave the aimed compound.

Molecular formula: C₂₈ H₄₂ N₄ O₄ S₃ ; SIMS:m/z=617[M+Na]⁺, 595[M+H]⁺,379, 346, 248, 245

Example 11 ##STR24##

25 mg of compound 2 was dissolved in 0.5 ml of dimethylformamide. 5.5 mgof N-hydroxysuccinimide and 9.9 mg of 1,3-dicyclohexylcarbodiimide wereadded to the mixture under ice-cooling, followed by stirring for 1 hour.Methylamine hydrochloride (14.6 mg)/dimethylformamide (1.0 ml) was addedto the reaction, which was stirred for 1 hour under ice-cooling and thenfor 16 hours at room temperature. Water was added to the reaction, whichwas extracted with ethyl acetate. The extract was purified by usingpreparative TLC to give 30 mg of compound 26, which was further purifiedby preparative HPLC.

Molecular formula:C₂₈ H₄₂ N₄ O₄ S₃ ; SIMS:m/z=579[M+H]⁺ ; ¹H-NMR(CDCl₃):0.90 ppm(3H, t, J=7.1 Hz), 1.30 ppm(3H, s), 1.30 ppm(3H,s), 1.30-1.38 ppm(4H, m), 1.51 ppm(3H, s), 1.57-163 ppm (2H, m), 2.63ppm(3H, s), 2.80 ppm(3H, d, J=4.9 Hz), 2.87-3.03 ppm (2H, m), 2.95ppm(1H, dd, J=11.5 Hz, 4.4 Hz), 3.14 ppm(1H, dd, J=11.5 Hz, 7.3 Hz),3.15 ppm(1H, d, J=11.5 Hz), 3.24 ppm(1H, dd, J=11.5 Hz, 7.3 Hz), 3.35ppm(1H, ddd, J=7.3 Hz, 7.1 Hz, 4.4 Hz), 3.48 ppm(1H, dd, J=11.5 Hz, 8.8Hz), 3.56 ppm(1H, d, J=11.5 Hz), 3.68 ppm(1H, d, J=7.1Hz), 4.19 ppm(1H,d, J=9.5 Hz), 4.42 ppm(1H, br), 4.74 ppm(1H, ddd, J=9.5 Hz, 8.8 Hz, 7.3Hz), 6.71 ppm(1H, dd, J=7.6 Hz, 1.0 Hz), 6.84 ppm(1H, dd, J=8.1 Hz, 1.0Hz), 6.96 ppm(1H, br), 7.21 ppm(1H, dd, J=8.1 Hz, 7.6 Hz), 12.83 ppm(1H,br). ¹³ C-NMR(CDCl₃):14.05 ppm(q), 22.49 ppm(t), 23.51 ppm(q), 24.17 ppm(q), 24.83 ppm(q), 26.14 ppm(q), 31.87 ppm(t), 32.20 ppm(t), 35.16 ppm(t), 35.44 ppm(t), 36.85 ppm(t), 41.52 ppm(t), 45.42 ppm(s), 46.27 ppm(q), 72.98 ppm(d), 78.10 ppm(d), 78.15 ppm(d), 78.15 ppm(d) 79.47 ppm(d), 84.31 ppm(s), 115.39 ppm(d), 116.19 ppm(s), 121.44 ppm(d), 132.27ppm(d), 143.82 ppm(s), 159.72 ppm(s), 171.99 ppm(s), 175.26 ppm(s),179.36 ppm(s)

Example 12 ##STR25##

35 mg of compound 2 was dissolved in 1.0 ml of dimethylformamide. 10.6mg of dimethylamine hydrochloride and 57 mg of BOP reagent were added tothe mixture with stirring on ice. N,N-diisopropylethylamine (17mg)/benzene (1.0 ml) was then added to the reaction, stirred for 15minutes at the same temperature and then for 24 hours at roomtemperature. To the reaction was added water, and the mixture wasextracted with ethyl acetate. The extract was subjected to preparativeTLC to give compound 27 (15.5 mg), and the mixture was further purifiedby preparative HPLC.

Molecular formula:C₂₉ H₄₄ N₄ O₄ S₃ ; SIMS:m/z=593[M+H]⁺, 379,344

Example 13 ##STR26##

54 mg of compound 1 was dissolved in 7 ml of methanol. To the mixture, asolution of diazomethane in ether was added, and the mixture was allowedto react for 1 hour at room temperature. After the reaction, thereaction mixture was concentrated to dryness in vacuo, which wasisolated and purified by using preparative TLC and preparative HPLC togive compound 4 (7.6 mg) and 5 (5.0 mg).

Compound 4:

Molecular formula:C₂₈ H₃₉ N₃ O₄ S₃ Zn; SIMS m/z=664[M+Na]⁺, 642[M+H]⁺ ;

Compound 5:

Molecular formula:C₂₈ H₄₁ N₃ O₄ S₃ ; FAB MS m/z=580[M+H]⁺ ; ¹H-NMR(CDCl₃ :0.90 ppm(3H, t, J=7.1 Hz), 1.28 ppm(3H, s), 1.30 ppm(3H,s), 1.32-1.39 ppm(4H, m), 1.53 ppm(3H, s), 1.59-1.67 ppm (2H, m), 2.64ppm(3H, s), 2.92-3.02 ppm(4H, m), 3.13 ppm(1H, m), 3.21 ppm(1H, d,J=11.7 Hz), 3.22 ppm(1H, dd, J=11.2 Hz, 8.8 Hz), 3.37 ppm(1H, d, J=7.8Hz), 3.40 ppm(1H, dd, J=11.2 Hz, 8.8 Hz), 3.62 ppm(1H, d, J=11.7 Hz),4.15 ppm(1H, d, J=8.1 Hz), 4.75 ppm(1H, dt, J=8.1 Hz, 8.8 Hz), 6.70ppm(1H, dd, J=7.6 Hz, 1.2 Hz), 6.84 ppm (1H, dd, J=8.3 Hz, 1.2 Hz), 7.20ppm(1H, dd, J=8.3 Hz, 7.6 Hz). ¹³ C-NMR(CDCl₃):14.06 ppm, 22.48 ppm,22.51 0ppm, 23.66 ppm, 26.04 ppm, 31.92 ppm, 32.34 ppm, 34.67 ppm, 34.86ppm, 35.55 ppm, 40.62 ppm, 45.50 ppm, 46.61 ppm, 61.96 ppm, 74.59 ppm,78.55 ppm, 78.92 ppm, 83.71 ppm, 90.43 ppm, 115.49 ppm, 116.02 ppm,121.34 ppm, 132.15 ppm, 143.85 ppm, 160.38 ppm, 171.68 ppm, 174.67 ppm,181.71 ppm.

Example 14 ##STR27##

199 mg of compound 1 was dissolved in pyridine, and to the mixture,anhydrous acetic acid (2.5 ml) was added, and the mixture was allowed toreact for 16 hours at room temperature, and then, dried in vacuo. Then,compound 8 (16 5 mg) was obtained by using preparative TLC, which wasfurther purified by using preparative HPLC.

Molecular formula:C₃₁ H₄₃ N₃ O₆ S₃ ; SIMS:m/z=672[M+Na]⁺, 650[M+H]⁺ ; ¹H-NMR(CDCl₃):0.88 ppm(3H, t, J=7.1 Hz), 1.32 ppm(6H, s), 1.32-1.35ppm(4H, m), 1.51 ppm(3H, s), 1.58-1.62 ppm(2H, m), 2.09 ppm(3H, s), 2.29ppm(3H, s), 2.57 ppm(3H, s), 2.64-2.68 ppm(2H, m), 2.97-3.06 ppm(2H, m),3.20 ppm(1H, m), 3.21 ppm(1H, d, J=11.7 Hz), 3.37 ppm(1H, dd, J=11.2 Hz,8.8 Hz), 3.49 ppm(1H, dd, J=11.2 Hz, 9.8 Hz), 3.63 ppm(1H, d, J=11.7Hz), 4.50 ppm(1H, d, J=5.6 Hz), 4.90 ppm(1H, ddd, J=9.8 Hz, 8.8 Hz, 5.6Hz), 5.30 ppm(1H, d, J=6.3 Hz), 6.94 ppm(1H, dd, J=8.0 Hz, 1.2 Hz), 7.10ppm(1H, dd, J=7.8 Hz, 1.2 Hz), 7.31 ppm(1H, dd, J=8.0 Hz, 7.8 Hz).

Example 15 ##STR28##

Compound 8 (145 mg) was dissolved in methanol (6 ml), and to the mixturean aqueous solution of 1 N sodium hydroxide (3 ml) was added withcooling, and the mixture was allowed to react for 16 hours at roomtemperature. The reaction was neutralized with 6 N hydrochloric acidwith cooling and methanol was concentrated in vacuo. To the residue,saturated saline was added and extracted with ethyl acetate. Thereaction was dried over anhydrous sodium sulfate and evaporated in vacuoto give compound 2 (109 mg), which was purified by using preparativeHPLC.

Example 16 ##STR29##

Compound 8 (22 mg) was dissolved in methanol (3 ml), and to the mixture,0.5 N sodium hydroxide was added with stirring on ice followed bystirring for 15 minutes. The reaction was adjusted to pH 6 by 0.5 Nhydrochloric acid under ice-cooling, and the methanol was evaporated invacuo. To the concentrate was added water, and the resultant mixture wasextracted with dichloromethane, washed with water, dried over anhydroussodium sulfate, and then, the solvent was removed by evaporation to give21 mg of compound 3. For a sample for identifying structure and assay,the compound was further purified by using HPLC.

SIMS m/z=608[M+H]⁺ ; ¹ H-NMR(CDCl₃):0.90 ppm(3H, t, J=7.2 Hz), 1.33ppm(3H, s), 1.35 ppm(3H, s), 1.35-1.39 ppm(4H, m), 1.52 ppm(3H, s),1.60-1.64 ppm (2H, m), 2.16 ppm(3H, s), 2.58 ppm(3H, s), 2.89-3.04ppm(2H, m), 3.04 ppm(1H, dd, J=11.7 Hz, 7.6 Hz), 3.09 ppm(1H, dd, J=11.7Hz, 7.1 Hz), 3.16 ppm(1H, dd, J=11.2 Hz, 8.3 Hz), 3.23 ppm(1H, d, J=11.5Hz), 3.30 ppm(1H, ddd, J=7.6 Hz, 7.1 Hz, 6.8 Hz), 3.36 ppm (1H, dd,J=11.2 Hz, 8.8 Hz), 3.65 ppm(1H, d, J=11.5 Hz), 4.16 ppm (1H, d, J=8.5Hz), 4.78 ppm(1H, ddd, J=8.8 Hz, 8.5 Hz, 8.3 Hz), 5.36 ppm(1H, d, J=6.8Hz), 6.69 ppm(1H, dd, J=7.6 Hz, 1.2 Hz), 6.83 ppm(1H, dd, J=8.3 Hz, 1.2Hz), 7.19 ppm(1H, dd, J=8.3 Hz, 7.6 Hz). ¹³ NMR(CDCl₃):14.06 ppm(q),21.05 ppm(q), 22.52 ppm(t ), 23.34 ppm(q), 23.72 ppm(q), 24.87 ppm(q),31.92 ppm(t), 32.30 ppm(t), 34.61 ppm(t), 35.40 ppm(t), 35.50 ppm(t),40.78 ppm(t), 44.76 ppm(q), 44.95 ppm(s), 71.86 ppm(d), 77.95 ppm(d),78.73 ppm(d), 78.89 ppm(d), 83.89 ppm(s), 115.45 ppm(d), 116.14 ppm(s),121.24 ppm(d), 132.02 ppm (d), 143.89 ppm(s), 160.30 ppm(s), 170.53ppm(s), 171.45 ppm(s), 174.73 ppm(s), 180.51 ppm(s).

Example 17 ##STR30##

Compound 8 (3.6 mg) was dissolved in methanol (1.0 ml), and to thesolution was added a solution of diazomethane in ether. After 30 minutesat room temperature, the reaction was concentrated to dryness, and theresidue was purified by using preparative HPLC to give compound 10 (2.4mg).

Molecular formula:C₃₂ H₄₅ N₃ O₆ S₃ ; SIMS:m/z=664[M+H]⁺ ; ¹H-NMR(CDCl₃):0.89 ppm(3H, t, J=7.0 Hz), 1.31-1.34 ppm(4H, m), 1.34ppm(6H, s), 1.54 ppm(3H, s), 1.59-1.61 ppm(2H, m), 2.10 ppm(3H, s), 2.29ppm(3H, s), 2.56 ppm(3H, s), 2.65-2.69 ppm(2H, m), 3.01 ppm (1H, dd,J=11.5 Hz, 6.8 Hz), 3.04 ppm(1H, dd, J=11.5 Hz, 6.8 Hz), 3.12 ppm(1H, d,J=11.2 Hz), 3.21 ppm(1H, q, J=6.8 Hz), 3.37 ppm(1H, dd, J=11.2 Hz, 9.0Hz), 3.51 ppm(1H, dd, J=11.2 Hz, 10.0 Hz), 3.67 ppm(1H, d, J=11.2 Hz),3.78 ppm(3H, s), 4.52 ppm(1H, d, J=5.4 Hz), 4.90 ppm(1H, ddd, J=10.0 Hz,9.0 Hz, 5.4 Hz), 5.21 ppm(1H, d, J=6.3 Hz), 6.94 ppm(1H, dd, J=8.1 Hz,1.0 Hz), 7.10 ppm(1H, dd, J=7.6 Hz, 1.0 Hz), 7.30 ppm(1H, dd, J=8.1 Hz,7.6 Hz).

Example 18 ##STR31##

To compound 10 (21 mg) dissolved in methanol (0.5 ml), 28% aqueousammonia solution (60 mg) was added at room temperature, and left tostand for 17 hours at the same temperature, and then, 28% aqueousammonia solution (60 mg) was added thereto, and left to stand for 24hours. 1N-sodium hydroxide (0.3 ml) was added to the reaction at roomtemperature, and then, stirred for 5 hours. The reaction was neutralizedwith 0.5 N hydrochloric acid. After water was added thereto, the mixturewas extracted with dichloromethane, washed with water, dried overanhydrous sodium sulfate, and the solvent was removed by evaporation togive a crude product of compound 31 (17 mg). A sample for identifyingstructure and assay was obtained by further purification usingpreparative HPLC.

Molecular formula:C₂₇ H₄₀ N₄ O₃ S₃ ; FAB MS:m/z=587[M+Na]⁺, 565[M+H]⁺,379, 349, 316

Example 19 ##STR32##

To a solution of compound 10 (23 mg) in anhydrous tetrahydrofurane (0.5ml) was added a solution of lithium borohydride (5 mg ) in anhydroustetrahydrofurane with stirring on ice. The mixture was stirred for 10minutes at room temperature. 3 mg of lithium borohydride was addedthereto under ice-cooling, and the mixture was stirred for 2 hours atroom temperature. Acetone and water were added to the reaction, and themixture was neutralized with 0.5 N hydrochloric acid, eluted with ethylacetate, washed with water, dried over anhydrous sodium sulfate, andthen, the solvent was removed by evaporation. The residue was subjectedto preparative TLC to give 9 mg of compound 32, which was furtherpurified by HPLC and the structure thereof was identified.

Molecular formula: C₂₇ H₄₁ N₃ O₃ S₃ ; SIMS:m/z=574[M+Na]⁺, 552[M+H]⁺,379,349,303,248

Example 20

    __________________________________________________________________________                                          #STR33##                                                                      #STR34##                                Compound No.         R.sup.4       R.sup.2       R.sup.3'                     __________________________________________________________________________    17                   Methoxymethyl Methoxymethyl Methoxymethyl                  18 Methoxymethyl Methoxymethyl H                                              19 Methoxymethyl H Methoxymethyl                                              20 Methoxymethyl H H                                                        __________________________________________________________________________

To compound 1 (50 mg) dissolved in dichloromethane, N,N-diisopropylethylamine (17 μl) and chloromethyl methyl ether (MOMCl)(7.0 μl) were added and the mixture was stirred for 5 hours. Thereaction mixture was poured into ice water containing potassium hydrogensulfate, and extracted with dichloromethane. The organic layer waswashed with saturated saline, dried over anhydrous sodium sulfate, andthe solvent was removed by evaporation in vacuo. The residue wassubjected to silica gel column chromatography (eluted consecutively with20-50% ethyl acetate hexane, ethyl acetate and 10-20% methanol/ethylacetate) for purification. After further repurification with HPLC,compound 17 (18.0 mg) compound 18 (14.2 mg), compound 19 (1.9 mg) andcompound 20 (5.8 mg) were obtained.

Compound 17:

Molecular formula: C₃₃ H₅₁ N₃ O₇ S₃ ; ¹ H-NMR(CDCl₃): 0.88 ppm(3H, t,J=7.1 Hz), 1.32 ppm(3H, s), 1.32-1.34 ppm(4H, m), 1.34 ppm(3H, s), 1.54ppm(3H, s), 1.58-1.65 ppm(2H, m), 2.57-2.69 ppm(2H, m), 2.60 ppm(3H, s),2.90 ppm(1H, dd, J=11.2 Hz, 8.3 Hz), 3.03 ppm(1H, dd, J=11.2 Hz, 6.4Hz), 3.10 ppm(1H, m), 3.11 ppm(1H, d, J=11.5 Hz), 3.41 ppm(1H, dd,J=10.7 Hz, 8.8 Hz), 3.42 ppm(3H, s), 3.47 ppm(3H, s), 3.48 ppm(3H, s),3.55 ppm(1H, dd, J=10.7 Hz, 9.8 Hz), 3.66 ppm(1H, d, J=11.5 Hz), 3.88ppm(1H, d, J=6.6 Hz), 4.49 ppm(1H, d, J=5.4 Hz), 4.73 ppm(1H, d, J=6.6Hz), 4.98 ppm(1H, ddd, J=9.8 Hz, 8.8 Hz, 5.4 Hz), 5.00 ppm (1H, d, J=6.6Hz), 5.14 ppm(1H, d, J=6.6 Hz), 5.19 ppm(1H, d, J=6.6 Hz), 5.29 ppm(1H,d, J=5.9 Hz), 5.35 ppm(1H, d, J=5.9 Hz), 6.87 ppm(1H, d, J=7.8 Hz), 6.94ppm(1H, d, J=7.8 Hz), 7.21 ppm(1H, t, J=7.8 Hz).

Compound 18:

Molecular formula: C₃₁ H₄₇ N₃ O₆ S₃ ; ¹ H-NMR(CDCl₃): 0.89 ppm(3H, t,J=7.0 Hz), 1.28 ppm(3H, s) 1.28-1.34 ppm(4H, m), 1.32 ppm(3H, s), 1.54ppm(3H, s), 1.59-1.63 ppm(2H, m), 2.61 ppm(3H, s), 2.61-2.65 ppm(2H, m),2.84 ppm(1H, dd, J=11.7 Hz, 3.7 Hz), 3.10 ppm(1H, d, J=11.5 Hz), 3.22ppm(1H, dd, J=11.7 Hz, 6.8 Hz), 3.34 ppm(1H, m), 3.36 ppm(1H, dd, J=11.2Hz, 7.8 Hz), 3.47 ppm(1H, d, J=7.1 Hz), 3.47 ppm(3H, s), 3.48 ppm(3H,s), 3.55 ppm(1H, dd, J=11.2 Hz, 8.8 Hz), 3.64 ppm(1H, d, J=11.5 Hz),4.37 ppm(1H, d, J=8.5 Hz), 4.74 ppm(1H, br), 4.86 ppm(1H, ddd, J=8.8 Hz,8.5 Hz, 7.8 Hz), 5.16 ppm(2H, s), 5.29 ppm(1H, d, J=5.9 Hz), 5.35ppm(1H, d, J=5.9 Hz), 6.88 ppm(1H, d, J=7.8 Hz), 6.95 ppm(1H, d, J=7.8Hz), 7.23 ppm(1H, t, J=7.8 Hz).

Compound 19:

Molecular formula: C₃₁ H₄₇ N₃ O₆ S₃ ; ¹ H-NMR(CDCl₃): 0.90 ppm(3H, t,J=7.1 Hz), 1.33 ppm(3H, s) 1.34 ppm(3H, s), 1.34-1.37 ppm(4H, m), 1.52ppm(3H, s), 1.57-1.62 ppm(2H, m), 2.61 ppm(3H, s), 2.92-3.01 ppm(3H, m),3.09 ppm(1H, dd, J=11.7 Hz, 6.8 Hz), 3.12 ppm(1H, d, J=11.5 Hz), 3.19ppm(1H, dd, J=11.2 Hz, 8.8 Hz), 3.20 ppm(1H, m), 3.38 ppm(1H, dd, J=11.2Hz, 8.8 Hz), 3.44 ppm(3H, s), 3.49 ppm(3H, s),3.68 ppm(1H, d, J=11.5Hz), 3.83 ppm(1H, d, J=7.6 Hz), 4.14 ppm(1H, d, J=8.5 Hz), 4.77 ppm(1H,dt, J=8.5 Hz, 8.8 Hz), 4.83 ppm(1H, d, J=6.6 Hz), 5.07 ppm(1H, d, J=6.6Hz), 5.28 ppm(1H, d, J=6.1 Hz), 5.36 ppm(1H, d, J=6.1 Hz), 6.69 ppm(1H,d, J=7.3 Hz), 6.84 ppm(1H, d, J=7.0 Hz), 7.19 ppm(1H, dd, J=7.3 Hz, 7.0Hz), 13.35 ppm(1H, br).

Compound 20:

Molecular formula: C₂₉ H₄₃ N₃ O₅ S₃ ; ¹ H-NMR(CDCl₃): 0.90 ppm(3H, t,J=7.1 Hz), 1.31 ppm(3H, s) 1.34 ppm(3H, s), 1.34-1.38 ppm(4H, m), 1.55ppm(3H, s), 1.55-1.63 ppm(2H, m), 2.63 ppm(3H, s), 2.86-3.04 ppm(2H, m),2.93 ppm(1H, dd, J=11.7 Hz, 4.4 Hz), 3.13 ppm(1H, d, J=11.5 Hz), 3.14ppm(1H, dd, J=11.5 Hz, 8.1 Hz), 3.26 ppm(1H, dd, J=11.7 Hz, 7.1 Hz),3.42 ppm(1H, ddd, J=7.1 Hz, 6.6 Hz, 4.4 Hz), 3.46 ppm(1H, dd, J=11.5 Hz,8.8 Hz), 3.48 ppm(3H, s), 3.52 ppm(1H, d, J=6.6 Hz), 3.66 ppm(1H, d,J=11.5 Hz), 4.19 ppm(1H, d, J=9.5 Hz), 4.38 ppm(1H, br), 4.77 ppm(1H,ddd, J=9.5 Hz, 8.8 Hz, 8.1 Hz), 5.30ppm(1H, d, J=5.9 Hz), 5.35 ppm (1H,d, J=5.9 Hz),6.70 ppm(1H, dd, J=7.6 Hz, 1.2 Hz), 6.85 ppm(1H, dd, J=8.3Hz, 1.2 Hz), 7.21 ppm(1H, ddr J=8.3 Hz, 7.6 Hz), 12.84 ppm (1H, br).

Example 21

    __________________________________________________________________________                                          #STR35##                                                                      #STR36##                                Compound No.        R.sup.4       R.sup.2        R.sup.3'                     __________________________________________________________________________    21                  --CH.sub.2 (4-ClC.sub.6 H.sub.4)                                                            --CH.sub.2 (4-ClC.sub.6 H.sub.4)                                                             --CH.sub.2 (4-ClC.sub.6                                                       H.sub.4)                       22 --CH.sub.2 (4-ClC.sub.6 H.sub.4) H H                                       23 --CH.sub.2 (4-ClC.sub.6 H.sub.4) H --CH.sub.2 (4-ClC.sub.6 H.sub.4)      __________________________________________________________________________

To a suspension consisting of compound 1 (5 0 mg), potassium carbonate(220 mg), dichloromethane (80 μl), dimethylformamide (400 μl) was added4-chlorobenzylbromide (35 mg) at room temperature, and the mixture wasstirred for 20 hours. Dichloromethane was added to the reaction mixture,and the mixture was consecutively washed with an aqueous solution of 5%potassium hydrogen sulfate and saturated saline. The organic layer wasdried over anhydrous sodium sulfate and the solvent was removed byevaporation in vacuo. The resultant residue was subjected to silica gelcolumn chromatography (eluted consecutively with 20%-80% ethylacetate/hexane and ethyl acetate), and the partially purified compoundwas repurified by using HPLC to give compound 21 (7.2 mg), compound 22(6.0 mg) and compound 23 (2 3.2 mg).

Compound 21:

Molecular formula: C₄₈ H₅₄ Cl₃ N₃ O₄ S₃ ; ¹ H-NMR(CDCl₃): 0.87 ppm(3H,t, J=7.1 Hz), 1.26 ppm(3H, s), 1.30 ppm(3H, s), 1.30-1.33 ppm(4H, m),1.50 ppm(3H, s), 1.59-1.61 ppm(2H, m), 2.51 ppm(3H, s), 2.64 ppm(2H, dd,J=9.3 Hz, 6.6 Hz), 2.90 ppm(1H, dd, J=11.4 Hz, 8.5 Hz), 3.02 ppm(1H, dd,J=11.4 Hz, 6.5 Hz), 3.06 ppm(1H, d, J=11.2 Hz), 3.11 ppm(1H, ddd, J=8.3Hz, 7.6 Hz, 6.5 Hz), 3.42 ppm(1H, dd, J=10.8 Hz, 8.5 Hz), 3.53 ppm(1H,dd, J=10.8 Hz, 9.0 Hz), 3.59 ppm(1H, d, J=11.2 Hz), 3.71 ppm(1H, d,J=7.6 Hz), 4.39 ppm(1H, d, J=6.4 Hz), 4.44 ppm(1H, d, J=11.0 Hz, 4.91ppm(1H, ddd, J=9.0 Hz, 8.5 Hz, 6.4 Hz), 5.04 ppm(2H, s, 5.09 ppm(1H, d,J=1.0 Hz), 5.09 ppm(1H, d, J=12.5 Hz), 5.16 ppm(1H, d, J=12.5 Hz), 6.72ppm(1H, d, J=8.3 Hz), 6.85 ppm(1H, d, J=7.8 Hz), 7.20-7.37 ppm(13H, m).

Compound 22:

Molecular formula: C₃₄ H₄₄ ClN₃ O₄ S₃ ; ¹ H-NMR(CDCl₃): 0.90 ppm(3H, t,J=7.1 Hz), 1.28 ppm(3H, s) 1.31 ppm(3H, s), 1.34-1.38 ppm(4H, m), 1.52ppm(3H, s), 1.59-1.63 ppm(2H, m), 2.58 ppm(3H, s), 2.85 ppm(1H, dd,J=11.7 Hz, 4.6 Hz ), 2.89 ppm(1H, m), 3.00 ppm(1H, m), 3.10 ppm(1H, dd,J=11.7 Hz, 7.0 Hz), 3.10 ppm(1H, d, J=11.5 Hz), 3.13 ppm(1H, dd, J=11.2Hz, 7.8 Hz), 3.35 ppm(1H, ddd, J=7.0 Hz, 6.6 Hz, 4.6 Hz), 3.44 ppm(1H,dd, J=11.2 Hz, 8.8 Hz), 3.49 ppm(1H, d, J=6.6 Hz), 3.59 ppm(1H, d,J=11.5 Hz), 4.16 ppm(1H, d, J=9.3 Hz ), 4.36 ppm(1H, br), 4.75 ppm(1H,ddd, J=9.3 Hz, 8.8 Hz, 7.8 Hz), 5.14 ppm(1H, d, J=12.5 Hz), 5.20 ppm(1H,d, J=12.5 Hz), 6.70 ppm(1H, dd, J=7.6 Hz, 1.2 Hz), 6.85 ppm(1H, dd,J=7.3 Hz, 1.2 Hz), 7.20 ppm(1H, dd, J=7.6 Hz, 7.3 Hz), 7.29 ppm(2H, d,J=8.8 Hz), 7.33 ppm(2H, d, J=8.8 Hz), 12.87 ppm(1H, br). ¹³C-NMR(CDCl₃): 14.05 ppm(q), 21.94 ppm(q), 22.49 ppm(t), 23.29 ppm(q),26.02 ppm(q), 31.88 ppm(t), 32.19 ppm(t), 35.00 ppm(t), 35.44 ppm(t),36.59 ppm(t), 41.20 ppm(t), 45.66 ppm(s), 45.86 ppm(q), 66.33 ppm(t),72.96 ppm(d), 77.72 ppm(d), 78.08 ppm(d) 79.62 ppm(d), 83.67 ppm(s),115.38 ppm(d), 116.25 ppm(s), 121.38 ppm(d), 128.82 ppm (d), 129.51ppm(d), 132.16 ppm(d), 134.06 ppm(s), 134.33 ppm(s), 143.79 ppm(s),159.73 ppm(s), 171.76 ppm(s), 172.85 ppm(s), 178.26 ppm (s).

Compound 23:

Molecular formula: C₄₁ H₄₉ Cl₂ N₃ O₄ S₃ ; ¹ H-NMR(CDCl₃): 0.90 ppm(3H,t, J=7.1Hz), 1.29 ppm(3H, s), 1.32 ppm(3H, s), 1.34-1.40 ppm(4H, m),1.50 ppm(3H, s), 1.60-1.66 ppm(2H, m), 2.58 ppm(3H, s), 2.91-3.07ppm(4H, m), 3.08 ppm(2H, d, J=11.2 Hz), 3.16-3.22 ppm(2H, m), 3.35ppm(1H, dd, J=11.4 Hz, 8.8 Hz), 3.61 ppm(1H, d, J=11.2 Hz), 3.75 ppm(1H,d, J=8.0 Hz), 4.11 ppm(1H, d, J=8.8 Hz), 4.56 ppm(1H, d, J=11.2 Hz),4.75 ppm(1H, dt, J=8.3 Hz, 8.8 Hz), 5.09 ppm(1H, d, J=12.5 Hz), 5.14ppm(1H, d, J=11.2 Hz), 5.17 ppm(1H, d, J=12.5 Hz), 6.70 ppm(1H, d, J=7.6Hz), 6.84 ppm(1H, d, J=7.6 Hz), 7.21 ppm(1H, t, J=7.6 Hz), 7.25-7.36 ppm(8H, m), 13.62 ppm(1H, br).

Example 22 ##STR37##

To a solution consisting of compound 23 (19. 6 mg) methanol (0.7 ml) and1,4-dioxane (0. 2 ml) was added an aqueous solution of 1 N sodiumhydroxide (50 μl), and the mixture was stirred for 24 hours. To thereaction mixture, dichloromethane was added, and the mixture was washedwith an aqueous solution of 5% potassium hydrogen sulfate. The organiclayer was dried over anhydrous sodium sulfate and the solvent wasremoved by evaporation in vacuo. The resultant residue was subjected tosilica gel column chromatography(eluted consecutively withdichloromethane,1% methanol/dichloromethane and 3%methanol/dichloromethane) to give compound 6(14.6 mg).

Molecular formula: C₃₄ H₄₄ ClN₃ O₄ S₃ ; FAB MS m/z=690[M+H]⁺, 441,125; ¹H-NMR(CDCl₃): 0.90ppm(3H, t, J=7.1Hz), 1.32 ppm(3H, s), 1.34 ppm(3H, s),1.36-1.39 ppm(4H, m), 1.51 ppm(3H, s), 1.59-1.65 ppm(2H, m), 2.60ppm(3H, s), 2.93-3.08 ppm(4H, m), 3.17-3.24 ppm(2H, m), 3.18 ppm(1H, d,J=11.7 Hz), 3.37 ppm(1H, dd, J=11.2 Hz, 8.5 Hz), 3.62 ppm(1H, d, J=11.7Hz), 3.70 ppm(1H, d, J=8.1 Hz), 4.14 ppm(1H, d, J=8.5 Hz), 4.55 ppm(1H,d, J=11.2 Hz), 4.75 ppm(1H, q, J=8.5 Hz), 5.16 ppm(1H, d, J=11.2 Hz),6.70 ppm(1H, dd, J=7.6 Hz, 1.3 Hz), 6.84 ppm(1H, dd, J=8.3 Hz, 1.3 Hz),7.21 ppm (1H, dd, J=8.3 Hz, 7.6 Hz), 7.28 ppm(2H, d, J=8.8 Hz), 7.35ppm(2H, d, J=8.8 Hz). ¹³ C-NMR (CDCl₃): 14.06 ppm(q), 22.50 ppm(t),22.56 ppm(q) 23.50 ppm(q), 26.24 ppm(q), 31.93 ppm(t), 32.35 ppm(t),34.84 ppm(t), 34.93 ppm(t), 35.54 ppm(t), 40.69 ppm(t), 45.95 ppm(q),46.58 ppm(s), 74.69 ppm(t), 74.79 ppm(d), 78.32 ppm(d), 79.03 ppm(d),83.70 ppm(s), 88.34 ppm(d), 115.54 ppm(d), 115.97 ppm(s), 121.32 ppm(d),128.35 ppm (d), 128.77 ppm(d), 132.21 ppm(d), 133.03 ppm(s), 137.21ppm(s), 143.82 ppm(s), 160.44 ppm(s), 171.89 ppm(s), 175.17 ppm(s),181.28 ppm (s).

Example 23 ##STR38##

To a solution consisting of compound 6(12.5 mg), diethyl ether (0.4 ml)and methanol (0.4 ml) was added 0.2 M trimethylsilyldiazomethane/hexanesolution (0.1 ml), and the mixture was stirred for 30 minutes at roomtemperature. The reaction mixture was diluted with ethyl acetate, washedconsecutively with an aqueous solution of 5% potassium hydrogen sulfateand saturated saline. The organic layer was dried over anhydrous sodiumsulfate and the solvent was removed by evaporation in vacuo. The residuewas subjected to silica gel column chromatography (eluted consecutivelywith dichloromethane and 20%ethyl acetate/dichloromethane) and purifiedby using HPLC to give compound 13 (5.4 mg).

Molecular formula: C₃₅ H₄₆ ClN₃ O₄ S₃ ; ¹ H-NMR(CDCl₃): 0.90 ppm(3H, t,J=7.1 Hz), 1.32 ppm(3H, s), 1.35 ppm(3H, s), 1.35-1.47 ppm(4H, m), 1.50ppm(3H, s), 1.60-1.66 ppm(2H, m), 2.60 ppm(3H, s), 2.91-3.05 ppm(3H, m),3.10 ppm(1H, d, J=11.5 Hz), 3.11 ppm(1H, dd, J=11.5 Hz, 7.3 Hz), 3.19ppm(1H, dd, J=11.2 Hz, 8.5 Hz), 3.21 ppm(1H, m), 3.38 ppm(1H, dd, J=11.2Hz, 8.8 Hz), 3.66 ppm(1H, d, J=11.2 Hz), 3.74 ppm(3H, s), 3.76 ppm(1H,d, J=8.1 Hz), 4.13 ppm(1H, d, J=8.8 Hz), 4.59 ppm(1H, d, J=11.2 Hz),4.77 ppm(1H, dt, J=8.5 Hz, 8.8 Hz), 5.17 ppm(1H, d, J=11.2 Hz), 6.70ppm(1H, dd, J=7.6 Hz, 1.2 Hz), 6.84 ppm(1H, dd, J=8.3 Hz, 1.2 Hz), 7.21ppm(1H, dd, J=8.3 Hz, 7.6 Hz), 7.28 ppm(2H, d, J=8.5 Hz), 7.37 ppm(2H,d, J=8.5 Hz), 13.58 ppm(1H, br). ¹³ C-NMR(CDCl₃): 14.07 ppm(q), 21.99ppm(q), 22.52 ppm(t), 23.39 ppm (q), 26.30 ppm(q), 31.94 ppm(t), 32.35ppm(t), 34.90 ppm(t ), 34.90 ppm (t), 35.54 ppm(t), 41.42 ppm(t), 46.00ppm(q), 46.48 ppm(s), 52.75 ppm (q), 74.49 ppm(t), 74.88 ppm(d), 78.24ppm(d), 79.36 ppm(d), 83.98 ppm (s), 88.30 ppm(d), 115.52 ppm(d), 116.05ppm(s), 121.23 ppm(d), 128.29 ppm(d), 128.79 ppm(d), 132.1 ppm(s),132.88 ppm(d), 137.55 ppm (s), 143.80 ppm(s), 160.50 ppm(s), 171.63ppm(s), 173.59 ppm(s), 178.81 ppm(s),

Example 24 ##STR39##

To a mixture of compound 1 (50 mg), L-alanine methylester hydrochloride(17 mg), triethylamine (17 μl), and dimethylformamide (0.4 ml), diethylcyanophosphate (18 μl) was added at temperature, and the mixture wasstirred for 25 hours. The reaction mixture was diluted withdichloromethane, and washed with an aqueous solution of 5% potassiumhydrogen sulfate. The organic layer was dried over anhydrous sodiumsulfate and the solvent was removed by evaporation in vacuo. The residuewas purified by using silica gel column chromatography (elutedconsecutively with dichloromethane, 20-50% ethyl acetate/hexane, ethylacetate, and 10-20% methanol/ethyl acetate) to give compound 28 (34 mg).

Molecular formula: C₃₁ H₄₆ N₄ O₅ S₃ ; ¹ H-NMR(CDCl₃): 0.90 ppm(3H, t,J=7.1Hz), 1.32 ppm(3H, s) 1.35 ppm(3H, s), 1.35-1.39 ppm(4H, m), 1.40ppm(3H, d, J=7.0 Hz), 1.50 ppm(3H, m), 1.59-1.63 ppm(2H, m), 2.66ppm(3H, s), 2.84-3.04 ppm(3H, m), 3.14 ppm(1H, dd, J=11.5 Hz, 7.8 Hz),3.17 ppm(1H, d, J=11.5 Hz), 3.23 ppm(1H, dd, J=11.5 Hz, 7.1 Hz), 3.41ppm(1H, dt, J=7.1 Hz, 4.2 Hz), 3.47 ppm(1H, dd, J=11.5 Hz, 8.8 Hz), 3.54ppm(1H, d, J=11.5 Hz), 3.60 ppm(1H, d, J=7.1 Hz), 3.72 ppm(3H, s), 4.20ppm(1H, d, J=9.3 Hz), 4.54 ppm(1H, dq, J=7.6 Hz, 7.0 Hz), 4.75 ppm(1H,ddd, J=9.3 Hz, 8.8 Hz, 7.8 Hz), 6.70 ppm(1H, dd, J=7.6 Hz, 1.3 Hz), 6.84ppm(1H, dd, J=8.3 Hz, 1.3 Hz), 7.21 ppm(1H, dd, J=8.3 Hz, 7.6 Hz), 7.43ppm(1H, d, J=7.6 Hz)

Example 25 ##STR40##

To compound 28 (21.5 mg) dissolved in methanol (1.7 ml), an aqueoussolution (35 μl ) of 1N sodium hydroxide was added with stirring at roomtemperature. After 24 hours, an aqueous solution (17 μl) of 1N sodiumhydroxide was dropped into the mixture, and, after additional 24 hours,about 2/3 of methanol was removed by evaporation in vacuo. The residuewas then acidified with an aqueous solution of 5% potassium hydrogensulfate and extracted with dichloromethane. The organic layer was driedover anhydrous sodium sulfate, and the solvent was removed byevaporation in vacuo. The residue was subjected to silica gel columnchromatography (eluted consecutively with 30-50% ethyl acetate/hexane,ethyl acetate, and 10% methanol/ethyl acetate) to give compound 29 (14.7mg).

Molecular formula: C₃₀ H₄₄ N₄ O₅ S₃ ; ¹ H-NMR(CDCl₃):0.90 ppm(3H, t,J=7.1 Hz), 1.29 ppm(3H, s) 1.31 ppm(3H, s), 1.33-1.37 ppm(4H, m), 1.42ppm(3H, d, J=7.1 Hz), 1.54 ppm(3H, s), 1.59-1.63 ppm(2H, m), 2.61ppm(3H, s), 2.86-3.01 ppm(3H, m), 3.15 ppm(1H, dd, J=11.5 Hz, 7.6 Hz),3.15 ppm(1H, d, J=11.5 Hz), 3.25 ppm(1H, dd, J=11.5 Hz, 7.1 Hz), 3.44ppm(1H, ddd, J=7.3 Hz, 7.1 Hz, 4.4 Hz), 3.48 ppm(1H, dd, J=11.5 Hz, 8.5Hz), 3.59 ppm(1H, d, J=11.5 Hz), 3.61 ppm(1H, d, J=7.3 Hz), 4.18 ppm(1H,d, J=9.5 Hz), 4.73 ppm(1H, dq, J=7.5 Hz, 7.1 Hz), 4.73 ppm(1H, ddd,J=9.5 Hz, 8.5 Hz, 7.6 Hz), 6.72 ppm(1H, dd, J=7.6 Hz, 1.2 Hz), 6.85ppm(1H, dd, J=8.3 Hz, 1.2 Hz), 7.22 ppm(1H, dd, J=8.3 Hz, 7.6 Hz), 7.55ppm(1H, d, J=7.5 Hz). ¹³ C-NMR(CDCl₃):14.05 ppm(q), 17.90 ppm(q), 22.49ppm(t), 23.87 ppm (q), 24.29 ppm(q), 24.89 ppm(q), 31.87 ppm(t), 32.15ppm(t), 35.12 ppm (t), 35.36 ppm(t), 36.64 ppm(t), 41.38 ppm(t), 45.54ppm(s), 46.06 ppm (q), 48.14 ppm(s), 72.89 ppm(d), 77.93 ppm(d), 78.19ppm(d), 79.30 ppm (d), 84.23 ppm(s), 115.60 ppm(d), 116.16 ppm(s),121.58 ppm(d), 132.39 ppm(d), 143.83 ppm(s), 159.57 ppm(s), 172.50ppm(s), 174.66 ppm (s), 174.96 ppm(s), 179.33 ppm(s).

Example 26 ##STR41##

To a mixture of compound 1 (10 mg), ethanolamine hydrochloride (2.0 mg),triethylamine (5.0 μl), dimethylformamide (0.3 ml) and dichloromethane(0.1 ml), diethyl cyanophosphate (3.0 μl) was added at room temperature.After 24 hours, ethanol amine (2 μl) and diethyl cyanophosphate (3.0 μl)were added to the reaction. After 5 hours, ethanol amine (2 μl) anddiethyl cyanophosphate (3.0 μl) were added to the mixture, and themixture was stirred for 20 hours. The reaction mixture was diluted withethyl acetate, and washed consecutively with an aqueous solution of 5%potassium hydrogen sulfate, an aqueous solution of saturated sodiumhydrogen carbonate and saturated saline. The organic layer was driedover anhydrous sodium sulfate and the solvent was removed by evaporationin vacuo. The residue was purified with silica gel column chromatography(eluted consecutively with dichloromethane and 5%methanol/dichloromethane), and further purified by using HPLC to givecompound 30 (4.5 mg).

Molecular formula: C₂₉ H₄₄ N₄ O₄ S₃ ; ¹ H-NMR(CDCl₃):0.90 ppm(3H, t,J=7.1 Hz), 1.30 ppm(6H, s), 1.34-1.49 ppm(4H, m), 1.58 ppm(3H, s),1.59-1.61 ppm(2H, m), 2.63 ppm(3H, s), 2.89-3.02 ppm(3H, m), 3.11ppm(1H, d, J=11.7 Hz), 3.15 ppm(1H, dd, J=11.5 Hz, 7.3 Hz), 3.27 ppm(1H,dd, J=11.7 Hz, 7.3 Hz), 3.32-3.46 ppm(3H, m), 3.50 ppm(1H, dd, J=11.5Hz, 8.5 Hz), 3.62-3.65 ppm(2H, m), 3.64 ppm(1H, d, J=11.7 Hz), 3.80ppm(1H, d, J=7.6 Hz), 4.17 ppm(1H, d, J=9.8 Hz), 4.55 ppm(1H, br), 4.72ppm(1H, ddd, J=9.8 Hz, 8.5 Hz, 7.3 Hz), 6.72 ppm(1H, dd, J=7.6 Hz, 1.3Hz), 6.84 ppm(1H, dd, J=8.3 Hz, 1.3 Hz), 7.22 ppm(1H, dd, J=8.3 Hz, 7.6Hz), 7.45 ppm(1H, br). ¹³ C-NMR(CDCl₃):14.05 ppm(q), 22.50 ppm(t), 22.92ppm(q), 4.12 ppm (q), 25.11 ppm(q), 31.88 ppm(t), 32.20 ppm(t), 35.29ppm(t, 35.40 ppm (t), 36.90 ppm(t), 41.90 ppm(t), 42.31 ppm(t), 45.36ppm(s), 46.44 ppm (q), 62.27 ppm(t), 72.99 ppm(d), 77.94 ppm(d), 78.48pm(d), 79.39 ppm (d), 84.40 ppm(s), 115.40 ppm(d), 116.17 ppm(s), 121.57ppm(d), 132.37 ppm(d), 143.86 ppm(s), 159.61 ppm(s), 172.28 ppm(s),175.68 ppm (s), 180.23 ppm(s).

The compounds of the formula (I), which are obtained in the same manneras described in the examples stated above, are shown in Tables 3-7. Thetables include the compounds obtained in the foregoing references andexamples.

                  TABLE 3                                                         ______________________________________                                        No.  R.sup.1        R.sup.2      R.sup.3'                                     ______________________________________                                        1    COO.sup.-  (Zn.sup.2+)                                                                       --           H                                              2 COOH H H                                                                    3 COOH H Ac                                                                   4 COO.sup.-  (Zn.sup.2+) -- Me                                                5 COOH H Me                                                                   6 COOH H CH.sub.2 (4-ClC.sub.6 H.sub.4)                                       7 COOH Me H                                                                   8 COOH Ac Ac                                                                  9 COONa H H                                                                   10 COOMe Ac Ac                                                                11 COOMe CH.sub.2 (4-ClC.sub.6 H.sub.4) H                                     12 COOMe H H                                                                  13 COOMe H CH.sub.2 (4-ClC.sub.6 H.sub.4)                                     14 COOMe Me H                                                                 15 COOMe Me MTPA                                                              16 COOMe Me ═O                                                            17 COOCH.sub.2 OMe CH.sub.2 OMe CH.sub.2 OMe                                  18 COOCH.sub.2 OMe CH.sub.2 OMe H                                             19 COOCH.sub.2 OMe H CH.sub.2 OMe                                             20 COOCH.sub.2 OMe H H                                                        21 COOCH.sub.2 (4-ClC.sub.6 H.sub.4) CH.sub.2 (4-ClC.sub.6 H.sub.4)                                          CH.sub.2 (4-ClC.sub.6 H.sub.4)                 22 COOCH.sub.2 (4-ClC.sub.6 H.sub.4) H H                                      23 COOCH.sub.2 (4-ClC.sub.6 H.sub.4) H CH.sub.2 (4-ClC.sub.6 H.sub.4)                                         24 CONMeO.sup.-  (Zn.sup.2+) -- H                                             25 CONMeOH H H                                26 CONHMe H H                                                                 27 CONMe.sub.2 H H                                                            28 CONHCHMeCOOMe H H                                                          29 CONHCHMeCOOH H H                                                           30 CONHCH.sub.2 CH.sub.2 OH H H                                               31 CONH.sub.2 H H                                                             32 CH.sub.2 OH H H                                                            33 CO--Cys--OH H H                                                            34 CO--Cys--OMe Me H                                                          35 CO--Gly--OH H H                                                            36 CO--Gly--OH H Me                                                           37 CO--Gly--OH Me H                                                           38 CO--Gly--OMe H H                                                           39 CO--Gly--OMe H Me                                                          40 CO--Gly--OMe Me H                                                          41 CO--Ser--OH H H                                                            42 CO--Ser--OMe H H                                                           43 CONH.sub.2 Et H                                                          ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        No.      R.sup.1           R.sup.2  R.sup.3'                                  ______________________________________                                        44       CONH.sub.2        iPr      H                                           45 CONH.sub.2 Me H                                                            46 CONHCH.sub.2 CH.sub.2 NH.sub.2 H H                                         47 CONHCH.sub.2 CH.sub.2 NHMe H H                                             48 CONHCH.sub.2 CH.sub.2 OMe H H                                              49 CONHCH.sub.2 CH.sub.2 SMe H H                                              50 CONHMe CHF.sub.2 H                                                         51 CONHMe Et H                                                                52 CONHMe Me H                                                                53 CONHMe Pr H                                                                54 CONMeOEt Et H                                                              55 CONMeOH CHF.sub.2 H                                                        56 CONMeOH Et H                                                               57 CONMeOH iPr H                                                              58 CONMeOH Me H                                                               59 CONMeOMe Me H                                                              60 CONMeOMe Me Me                                                             61 COOCH.sub.2 (2,3-Cl.sub.2 C.sub.6 H.sub.3) H H                             62 COOCH.sub.2 (2,3-Cl.sub.2 C.sub.6 H.sub.3) H Me                            63 COOCH.sub.2 (2,3-Cl.sub.2 C.sub.6 H.sub.3) Me H                            64 COOCH.sub.2 (2,4-Cl.sub.2 C.sub.6 H.sub.3) H H                             65 COOCH.sub.2 (2,4-Cl.sub.2 C.sub.6 H.sub.2) H Me                            66 COOCH.sub.2 (2,4-Cl.sub.2 C.sub.6 H.sub.3) Me H                            67 COOCH.sub.2 (2-ClC.sub.6 H.sub.4) H H                                      68 COOCH.sub.2 (3,4-Cl.sub.2 C.sub.6 H.sub.2) H H                             69 COOCH.sub.2 (3-ClC.sub.6 H.sub.4) H H                                      70 COOCH.sub.2 (4-FC.sub.6 H.sub.4) H H                                       71 COOCH.sub.2 (4-FC.sub.6 H.sub.4) H Me                                      72 COOCH.sub.2 (4-FC.sub.6 H.sub.4) Me H                                      73 COOCH.sub.2 (4-MeC.sub.6 H.sub.4) H H                                      74 COOCH.sub.2 (4-MeC.sub.6 H.sub.4) H Me                                     75 COOCH.sub.2 (4-MeC.sub.6 H.sub.4) Me H                                     76 COOCH.sub.2 CH.sub.2 F H H                                                 77 COOCH.sub.2 CH.sub.2 F H Me                                                78 COOCH.sub.2 CH.sub.2 F Me H                                                79 COOCH.sub.2 OMe H Me                                                       80 COOCH.sub.2 OMe Me H                                                       81 COOCH.sub.2 Ph H H                                                         82 COOCH.sub.3 Ph H Me                                                        83 COOCH.sub.2 Ph Me H                                                        84 COOCH.sub.2 SMe H H                                                      ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        No.     R.sup.1      R.sup.2        R.sup.3'                                  ______________________________________                                        85      COOCH.sub.2 SMe                                                                            H              Me                                          86 COOCH.sub.2 SMe Me H                                                       87 COOCHF.sub.2 CHF.sub.2 H                                                   88 COOEt CHF.sub.2 Me                                                         89 COOEt Et Et                                                                90 COOEt Et H                                                                 91 COOEt H H                                                                  92 COOEt H Me                                                                 93 COOEt Me H                                                                 94 COOH CH.sub.2 (2-ClC.sub.6 H.sub.4) H                                      95 COOH CH.sub.2 (3,4-Cl.sub.2 C.sub.6 H.sub.3) H                             96 COOH CH.sub.2 (3-ClC.sub.6 H.sub.4) H                                      97 COOH CH.sub.2 (4-MeC.sub.6 H.sub.4) H                                      98 COOH CH.sub.2 (4-MeC.sub.6 H.sub.4) Me                                     99 COOH CH.sub.2 CH.sub.2 F H                                                 100 COOH CH.sub.2 CH.sub.2 F Me                                               101 COOH CH.sub.2 OEt H                                                       102 COOH CH.sub.2 OMe H                                                       103 COOH CH.sub.2 Ph H                                                        104 COOH CH.sub.2 Ph Me                                                       105 COOH CHF.sub.2 Ac                                                         106 COOH CHF.sub.2 COCF.sub.3                                                 107 COOH CHF.sub.2 COEt                                                       108 COOH CHF.sub.2 COiPr                                                      109 COOH CHF.sub.2 COPh                                                       110 COOH CHF.sub.2 COPr                                                       111 COOH CHF.sub.2 Et                                                         112 COOH CHF.sub.2 H                                                          113 COOH CHF.sub.2 iPr                                                        114 COOH CHF.sub.2 Me                                                         115 COOH CHF.sub.2 Pr                                                         116 COOH COEt COEt                                                            117 COOH COEt H                                                               118 COOH COiPr COiPr                                                          119 COOH COiPr H                                                              120 COOH CONHMe H                                                             121 COOH CONHMe Me                                                            122 COOH COOCH.sub.2 Ph H                                                     123 COOH COOCH.sub.2 Ph Me                                                    124 COOH COOEt H                                                              125 COOH COOEt Me                                                           ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        No.       R.sup.1   R.sup.2    R.sup.3'                                       ______________________________________                                        126       COOH      COOMe      H                                                127 COOH COOMe Me                                                             128 COOH COPh COPh                                                            129 COOH COPh H                                                               130 COOH Et H                                                                 131 COOH Et Me                                                                132 COOH H ═O                                                             133 COOH H CH.sub.2 (2-ClC.sub.6 H.sub.4)                                     134 COOH H CH.sub.2 (3-ClC.sub.6 H.sub.4)                                     135 COOH H CH.sub.2 (4-MeC.sub.6 H.sub.4)                                     136 COOH H CH.sub.2 CH.sub.2 F                                                137 COOH H CH.sub.2 Ph                                                        138 COOH H COBu                                                               139 COOH H COCF.sub.3                                                         140 COOH H COEt                                                               141 COOH H COiPr                                                              142 COOH H CONHMe                                                             143 COOH H COOCH.sub.2 Ph                                                     144 COOH H COOEt                                                              145 COOH H COOMe                                                              146 COOH H COPh                                                               147 COOH H COPr                                                               148 COOH H Et                                                                 149 COOH H iPr                                                                150 COOH H Pr                                                                 151 COOH iPr H                                                                152 COOH iPr Me                                                               153 COOH Me Ac                                                                154 COOH Me CH.sub.2 (4-MeC.sub.6 H.sub.4)                                    155 COOH Me CH.sub.2 CH.sub.2 F                                               156 COOH Me CH.sub.2 Ph                                                       157 COOH Me COCF.sub.3                                                        158 COOH Me COEt                                                              159 COOH Me COiPr                                                             160 COOH Me CONHMe                                                            161 COOH Me COOCH.sub.2 Ph                                                    162 COOH Me COOEt                                                             163 COOH Me COOMe                                                             164 COOH Me COPh                                                              165 COOH Me COPr                                                              166 COOH Me Et                                                                167 COOH Me iPr                                                               168 COOH Me Me                                                                169 COOH Pr H                                                               ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        No.     R.sup.1   R.sup.2      R.sup.3'                                       ______________________________________                                        170     COOiPr    H            H                                                171 COOiPr H Me                                                               172 COOiPr iPr H                                                              173 COOiPr Me H                                                               174 COOMe CH.sub.2 (4-MeC.sub.6 H.sub.4) H                                    175 COOMe CH.sub.2 CH.sub.2 F H                                               176 COOMe CH.sub.2 Ph H                                                       177 COOMe COCF.sub.2 COCF.sub.3                                               178 COOMe COEt COEt                                                           179 COOMe CONHMe H                                                            180 COOMe CONHMe Me                                                           181 COOMe COOCH.sub.2 Ph H                                                    182 COOMe COOCH.sub.2 Ph Me                                                   183 COOMe COOEt H                                                             184 COOMe COOEt Me                                                            185 COOMe COOMe H                                                             186 COOMe COOMe Me                                                            187 COOMe COPh COPh                                                           188 COOMe Et H                                                                189 COOMe H CH.sub.2 (3,4-Cl.sub.2 C.sub.6 H.sub.3)                           190 COOMe H CH.sub.2 (4-MeC.sub.6 H.sub.4)                                    191 COOMe H CH.sub.2 CH.sub.2 F                                               192 COOMe H CH.sub.2 Ph                                                       193 COOMe H CONHMe                                                            194 COOMe H COOCH.sub.2 Ph                                                    195 COOMe H COOEt                                                             196 COOMe H COOMe                                                             197 COOMe H Et                                                                198 COOMe H iPr                                                               199 COOMe iPr H                                                               200 COOMe Me CH.sub.2 OMe                                                     201 COOMe Me CONHMe                                                           202 COOMe Me COOCH.sub.2 Ph                                                   203 COOMe Me COOEt                                                            204 COOMe Me COOMe                                                            205 COOMe Me Me                                                             ______________________________________                                    

EXPERIMENT 1. Antibacterial Activities

(1) Methods

For bacteria, minimum Inhibitory Concentration (MIC, μg/mL) wasdetermined using the agar dilution method designated by the Society forChemotherapy in Japan. For Mycoplasma, the liquid dilution method wasused for determining MIC. Serpulina and Clostridium among the bacteriatested were anaerobically cultured using Gas-pack method. Determinationof MIC was conducted using the media having the following compositionsdepending on the bacteria tested.

PPLO medium (Difco) containing 12% horse serum and 1% glucose was usedfor Mycoplasma (M.) gallisepticum; a medium consisting of Hanks solutionwith 0.5% lactalbumin, 10% horse serum, 5% of 25% yeast extract, and 1%glucose for M. hyopneumoniae; and PPLO medium (Difco) containing 20%horse serum, 10% of 25% yeast extract, and 1% glucose for M. pneumoniae.Those media also contained 0.025% phenol red in order to enabling todetect the growth of the bacteria according to color changes. MIC valueswere determined after culturing at 37° C. for 120-168 hours.

Anaerobic culture was performed using Gas-pack method with TrypticaseSoy agar (TSA, BBL) containing 5% defibrinated sheep blood for Serpulinahyodysenteriae, and with GAM medium (Nissui) for Clostridiumperfringens. After culturing Serpulina hyodysenteriae at 37° C. for96-120 hours and Clostridium perfringens at 37° C. for 24 hours, MICswere determined.

Antibacterial activities for Branhamella catarrhalis were determinedaccording to the method of determining Minimum Growth InhibitoryConcentration designated by the Society for Chemotherapy in Japan. Thus,sensitivity-disc medium (Nissui) was used as a sensitivity determinationmedium, to which about 1 μl of 10⁶ CFU/ml of the bacterial suspensionwas inoculated.

Other bacteria were cultured at 37° C. for 20-24 hours using MHA for adetermination medium before determination of MIC.

(2) Results

The antibacterial activities of the compounds tested was shown in Table8.

Especially, low MICs of compounds 7, 8, 12, 18, 20 and 28 to Mycopasmaswere obserbed.

In addition, compounds 7, 18 and 20 showed the activity to Serpulina(Treponema) hyodysenteriae and Clostridium perflingens as well.

                                      TABLE 8                                     __________________________________________________________________________    In vitro determination results (MIC: μg/ml)                                   Mycoplasma                                                                    Mp    Mg   Mh                                                                 Mac S6 ST-11                                                                 No. (human) (avian) (swine) Antibacterial activities to other bacteria      __________________________________________________________________________    7  0.1   1.56 0.1  S. hyodysenteriae ATCC 27164                                                                 1.56                                              S. aureus FDA 209P 1.56                                                       C. perfringens ATCC 13124 0.1                                             9 0.00625 0.2 0.025 B. catarrhalis A 25238 3.13                                   C. trachmatis D/UW-3/CX 16                                                    E. avium A 14025 1.56 (50)                                                12 ≦0.00625 0.2 ≦0.025                                          18 0.025 1.56 0.05                                                            19 0.39 6.25 1.56                                                             20 ≦0.00625 0.1 0.025 S. hyodysenteriae ATCC 27164 1.56                    C. perfringens ATCC 13124 3.13                                            22 0.39 >3.13 0.1                                                             24 0.025 0.39 0.1                                                             26 0.78 6.25 6.25                                                             27 0.78 6.25 6.25                                                             28 0.05 0.39 0.1                                                              30 0.2 0.78 0.39                                                              31 0.2 0.39 0.2                                                               32 0.025 0.39 0.05                                                          __________________________________________________________________________     Mp:M. pneumoniae, Mg: M. gallisepticum, Mh: M. hypneumoniae              

2. In vivo Evaluation Test of Compound 9 for M. gallisepticum

(1) Methods

Eight-day old chickens (layer type, 6 chickens/group) were inoculatedwith 2.5×10² CFU of M. gallisepticum (field-isolated, macrolideresistant strain) per chicken in their right air sacs, and immediatelyafter inoculation, they forcibly received a single dose of 25, 50, 100mg/kg of compound 9.

Autopsy of the chickens was performed on 7th day after infection and theantibacterial effect was determined according to the incidence of theair-sac lesions and severity thereof.

Chlortetracycline formulation to be used only for animals was used as acontrol agent. The compounds and control agent were used after dissolvedin 3% arabic gum.

(2) Results

As shown in Table 9, the administration of the compound resulted in thedecrease in the number of chickens having air-sac lesions, whichconfirmed effectiveness of the compound.

                                      TABLE 9                                     __________________________________________________________________________                            The                                                        The number                                                                 Compound   number of air-sac lesions                                          No. or   of chickens (the number of                                           Drug Dose (mg × Administration chickens having chickens)**            Name  times)                                                                              method tested                                                                             lesions                                                                            (-)                                                                             (+)                                                                             (++-+++)                                     __________________________________________________________________________    9     25 × 1                                                                        Orally 6    2    4 0 2                                               50 × 1 Orally 6 2 4 1 1                                                 100 × 1 Orally 6 1 5 0 1                                               CTC* 50 × 1 Orally 6 3 3 0 3                                             100 × 1 Orally 6 1 5 0 1                                                200 × 1 Orally 6 1 5 0 1                                             Infected, not-administered control                                                               6    6    0 4 2                                              Not-infected, not-administered control 6 0 6 0 0                            __________________________________________________________________________     *CTC: chlortetracycline                                                       **(-): non, (+): slight, (++)-(+++): moderate to severe.                 

3. Antifungal Activities

Method:

Antifungal activities of the compounds to Candida albicans (strainCa-15) and Asperilhm fumigatus (strain MA), representative pathogenicfungi, were determined by micro-liquid dilution method using 96-wellmicroplates. A fungal suspension stored under freezing was adjusted to1×1 O⁵ CFU/ml by medium, of which 100 μl portions were charged into eachwell. The compound of the present invention was dissolved indimethylsulfoxide (DMSO). Using the resultant solution, 2-fold dilutionseries were prepared together with the fungal suspension charged intothe wells. YNB medium and RPMI medium were used. After cultured for 24hours at 30° C., evaluation was performed by detecting the turbidity(wave length: 595 nm) using microplate reader.

(2) Results

                  TABLE 10                                                        ______________________________________                                        Antifungal activities: IC.sub.50 (μg/ml)                                               Candida albicans                                                                            Aspergillus fumigatus                               Compound No.                                                                              YNB    RPMI       YNB  RPMI                                       ______________________________________                                        2           6.3    1.6        3.2  0.2                                          9 6.3 1.6 3.2 0.2                                                             20 >100 3.2 >100 >100                                                         25 50 6.3 50 0.2                                                            ______________________________________                                    

Strong antifungal activities of the compounds stated above were observedin vitro.

4. Growth Inhibition Activities to Avian Coccidial Protozoa

The kidneys of SPF new born chicken was sterilely removed, which wastreated with trypsin and the resultant primary kidney cells werecultured in 24-well plate. Monolayer culture after cultured for 3 dayswas treated simultaneously with compounds adjusted to predeterminedconcentration and sporozoit obtained by artificial excystation, whichwas cultured a 40° C. Premature and mature schizonts were observed undermicroscope, and the growth inhibition activities (suppression rate) foreach treatment group were calculated at a proportion of the number ofschizonts for the sporozoit-inoculated, compound-untreated control group(100%). As shown in Table 11, out of several compounds which showed saidactivities, compounds 7 and 19 especially showed strong growthinhibition activities. Concentrations showing growth inhibition effectswas 156 μg/ml. The compounds did not show any cytotoxicity to theprimary kidney culture cells at the same concentration.

                  TABLE 11                                                        ______________________________________                                        In vitro growth inhibition activities* to an avian coccidium,                   Eimeria tenella                                                                               Effective dose                                                                              Cytotoxicity                                    Compound No. (μg/ml) (μg/ml)                                          ______________________________________                                        2             2.5           10                                                  3 10 >10                                                                      7 0.156 10                                                                    10 >10 >10                                                                    11 10 >10                                                                     12 0.625 >10                                                                  13 >10 >10                                                                    18 10 >10                                                                     19 0.156 10                                                                   20 0.625 >10                                                                  21 >10 >10                                                                    22 2.5 >10                                                                    28 10 >10                                                                     30 2.5 >10                                                                    31 -- 10                                                                      32 10 >10                                                                   ______________________________________                                         *Growth inhibition activities: regarded more than 80% of suppression rate     as effective.                                                            

5. Effect of Suppression of Reaction to Mitogen of Mice Kidney Cells, invitro

To each well of 96 well microtiter plate were added 5×10⁵ spleen cellsof BDF1 mice suspended in 0.1 ml of RPMI 1640 medium supplemented with10% bovine fetus serum (supplemented with 2 mM sodium hydrogencarbonate, 50 Units/ml of penicillin, 50 μg/ml of streptomycin, and5×10⁻⁵ M of 2-mercaptoethanol), and then, 5 μg/ml of Concanavalin A (ConA) or 10 μg/ml of lypopolysaccaride (LPS) and various concentrations oftest substance, of which final volume was adjusted to 0.2 ml. Testsubstances were dissolved in dimethylsulfoxide (DMSO), diluted with RPMI1640 stated above, and added to the wells so that final concentration ofthe substances becomes less than 10000 ng/ml. The 96-well microtiterplates were cultured at 37° C. for 3 days in an incubator which wasmaintained at the humidity of 100%, 5% of carbon dioxide and 95% of air.After cultivation is completed, 25 ml of 6 mg/ml solution of MTT{3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazoliumbromide} (Sigma) isadded to each well, which is cultured at 37° C. for 4 days under thesame conditions. After cultivation is completed, formazan formed isdissolved by adding 50 μl of a solution of 20% dodecyl sodium sulfonate(SDS) in 0.02N hydrochloric acid and kept standing at 37° C. for 24hours. The absorbance (OD) of the formazan which was produced inproportion to the number of alive cells, was determined by usingimmunoreader equipped with 570 nm filter (see The Journal ofImmunological Methods, Vol.65, 55-63, 1983).

Cell growth inhibition rate and IC₅₀ value were calculated according tothe correlation between the concentration and the absorbance for eachsubstance.

The results are given in Tables 12 and 13.

                  TABLE 12                                                        ______________________________________                                        Suppressive effects of the following compounds on the                           reaction to Concanavalin A of mice spleen cells                               Concentration                                                                            Suppression rate (%)                                             of compound                                                                            Compound  Compound  Compound                                                                              Compound                                   (ng/ml) 6 7 18 20                                                           ______________________________________                                        39                 10.0      14.7                                               78  16.5 25.7                                                                 156 0 60.7 75.5                                                               312 16.5 98.0 94.9 0                                                          625 87.4 100.8 98.6 102.0                                                     1250 98.6   101.0                                                             2500 102.5   101.0                                                            IC.sub.50 values 399 129 101 506                                            ______________________________________                                    

                  TABLE 13                                                        ______________________________________                                        Suppressive effects of the following compounds on the                           reaction to lypopolusaccaride of mice spleen cells                            Concentration                                                                            Suppression rate (%)                                             of compound                                                                            Compound  Compound  Compound                                                                              Compound                                   (ng/ml) 6 7 18 20                                                           ______________________________________                                        39                 17.2      27.4                                               78  42.5 41.5                                                                 156 17.7 80.0 74.6                                                            312 24.4 98.6 93.4 2.1                                                        625 84.3 102.4 100.6 98.8                                                     1250 98.0   98.2                                                              2500 104.7   96.3                                                             IC.sub.50 values 368 84 84 380                                              ______________________________________                                    

As shown in Tables 11 and 12, compounds 6, 7, 18 and 20 suppressed thereactions to Con A and LPS of spleen cells.

EFFECT OF THE INVENTION

The compound represented by the formula (I) of the present invention hasespecially anti-mycoplasma acitivity, antibacterial activities,antifungal activities and immunosuppressive activities and is useful asan anti-mycoplasma agent, antibacterial agent, antifungal agent andimmunosuppressor.

We claim:
 1. A compound represented by the formula (I): ##STR42##wherein R¹ is (1) COOR⁴ wherein R⁴ is hydrogen atom, optionallysubstituted alkyl, optionally substituted aralkyl, optionallysubstituted heteroaryl or optionally substituted heteroarylalkyl; (2)CONR⁵ R⁶ wherein R⁵ and R⁶ are the same or different and representhydrogen atom, hydroxyl, optionally substituted alkyl, optionallysubstituted alkoxyl, optionally substituted aryl, optionally substitutedaralkyl, optionally substituted heteroaryl or optionally substitutedheteroarylalkyl, provided that when one of R⁵ and R⁶ is hydroxyl oroptionally substituted alkoxyl, the other is hydrogen atom, optionallysubstituted alkyl, optionally substituted aryl or optionally substitutedaralkyl; (3) Co--R⁷ --OR wherein R⁷ is α-amino acid residue and R ishydrogen atom or alkyl; or (4) CH₂ OR⁸ wherein R⁸ is hydrogen atom,optionally substituted alkyl, optionally substituted aralkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl, COR⁹wherein R⁹ is hydrogen atom, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl or optionally substituted heteroarylalkyl, COOR¹⁰ wherein R¹⁰is optionally substituted alkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl or optionallysubstituted heteroarylalkyl, or CONR¹¹ R¹² wherein R¹¹ and R¹² are thesame or different and represent hydrogen atom, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl or optionally substitutedheteroarylalkyl,R² is hydrogen atom, optionally substituted alkyl,optionally substituted aralkyl, optionally substituted heteroaryltoptionally substituted heteroarylalkyl, COR¹³ wherein R¹³ is hydrogenatom, optionally substituted alkyl, optionally substituted aryl,optionally substituted aralkyl, optionally substituted heteroaryl oroptionally substituted heteroarylalkyl, COOR¹⁴ wherein R¹⁴ is optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedaralkyl, optionally substituted heteroaryl or optionally substitutedheteroarylalkyl, or CONR¹⁵ R¹⁶ wherein R¹⁵ and R¹⁶ are the same ordifferent and represent hydrogen atom, optionally substituted alkyl,optionally substituted aryl, optionally substituted aralkyl, optionallysubstituted heteroaryl or optionally substituted heteroarylalkyl; abroken line ( - - - ) represents the presence or absence of a doublebond, provided that when it presents the presence of the double bond, R³is oxygen atom, or when it represents the absence of the double bond, R³is OR^(3') wherein R^(3') is hydrogen atom, optionally substitutedalkyl, optionally substituted aralkyl, optionally substitutedheteroaryl, optionally substituted heteroarylalkyl , COR¹⁷ wherein R¹⁷is hydrogen atom, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted heteroarylor optionally substituted heteroarylalkylt COOR¹⁸ wherein R¹⁸ isoptionally substituted alkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl or optionallysubstituted heteroarylalkyl, or CONR¹⁹ R²⁰ wherein R¹⁹ and R²⁰ are thesame or different and represent hydrogen atom, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl or optionally substitutedheteroarylalkyl, or a salt or metal chelate thereof, provided that themetal chelate is excluded when R¹ is COOH, R² is hydrogen atom and R³ isOH.
 2. A compound of claim 1 wherein R¹ is COOR⁴ wherein R⁴ is hydrogenatom, optionally substituted alkyl or optionally substituted benzyl, ora salt or metal chelate thereof.
 3. A compound of claim 1 wherein R¹ isCONR⁵ R⁶, wherein R⁵ and R⁶ are the same or different and representhydrogen atom, hydroxyl, alkoxyl, alkyl, carboxyalkyl,alkoxycarbonylalkyl or hydroxyalkyl, or a salt or metal chelate thereof.4. A compound of claim 1 wherein R² is hydrogen atom, optionallysubstituted alkyl, optionally substituted benzyl or alkanoyl, or a saltor metal chelate thereof.
 5. A compound of claim 1 wherein R³ is OR³ ',wherein R³ ' is hydrogen atom, optionally substituted alkyl, optionallysubstituted benzyl or alkanoyl.
 6. A medical drug comprising a compoundof claim 1 or a salt or metal chelete thereof, as an effectiveingredient.
 7. An animal drug comprising a compound of claim 1 or a saltor metal chelete thereof, as an effective ingredient.
 8. Ananti-mycoplasma agent comprising a compound of claim 1 or a salt ormetal chelete thereof, as an effective ingredient.
 9. An anticoccidalagent comprising a compound of claim 1 or a salt or metal cheletethereof, as an effective ingredient.
 10. An antibacterial agentcomprising a compound of claim 1 or a salt or metal chelete thereof, asan effective ingredient.
 11. An antifungal agent comprising a compoundof claim 1 or a salt or metal chelete thereof, as an effectiveingredient.
 12. An immunosuppressor comprising a compound of claim 1 ora salt or metal chelete thereof, as an effective ingredient.